Methods for assaying t-cell dependent bispecific antibodies

ABSTRACT

The present invention provides a cell-based assay for measuring T cell activation mediated by a T cell -dependent bispecific antibody (TDB). In some aspects, the assay is useful for detecting a TDB in a composition, quantitating the amount of TDB in a composition, determining the potency and/or specificity of a TDB, or determining if a population of cells expresses a target antigen. Compositions and kits are also contemplated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application62/286,862, filed Jan. 25, 2016, the contents of which are herebyincorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392036240SEQLIST.TXT,date recorded: Jan. 20, 2017, size: 1.7 KB).

FIELD OF THE INVENTION

The present invention provides methods for analyzing preparations ofmultispecific antibodies having an antigen binding fragment that binds aT cell receptor complex subunit, such as a CD3 subunit, and an antigenbinding fragment that binds a target antigen. In some embodiments, theinvention provides methods for detecting a TDB in a composition,quantitating the amount of TDB in a composition, determining the potencyand/or specificity of a TDB, or determining if a population of cellsexpresses a target antigen. Compositions and kits are also contemplated.

BACKGROUND OF THE INVENTION

T cell dependent bispecific antibodies (TDBs) are bispecific antibodiesdesigned to bind a target antigen expressed on a target cell and a Tcell receptor (TCR) complex subunit (e.g., CD3 subunit, such as CD3)expressed on a T cell. Binding of the bispecific antibody to theextracellular domains of both the target antigen and the TCR complexsubunit (TCS) results in T cell recruitment to target cells, leading toT cell activation and target cell depletion Certain combinations oftarget antigen-specific and TCS-specific (such as CD3-specific) antigenbinding fragments will be more effective than others for specificallyactivating T cells in the presence of target cells. Weakly activatingTDBs will have little therapeutic benefit. Non-specific activation of Tcells in the presence of off-target cells could lead to undesirablerelease of inflammatory cytokines. It is therefore desirable to assaythe degree and specificity of T cell activation mediated by various TDBsin order to support the development of safe and efficacious clinicaldrug candidates.

Optimal T cell activation assays should be accurate, precise, anduser-friendly, with short turnaround time and suitability for automationand high-throughput scaling. Several traditional bioassays areavailable, such as PBMC-based methods, FACS-based methods, and ELISA forsecreted cytokines. Unfortunately, many of these assays yield highlyvariable results and/or are time consuming. The novel TDB assaysdescribed herein use a cell-based approach with target cells andreporter T cells to detect T cell activation, and are validated byELISA-based bridging binding assays to detect simultaneous binding ofboth TDB antigen binding fragments to their targets.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

BRIEF SUMMARY

The invention provides methods for detecting T cell activation mediatedby a T cell dependent bispecific antibody (TDB), wherein the TDBcomprises a target antigen-binding fragment and a cell receptor complexsubunit (TCS)-binding fragment, and various uses thereof

In some embodiments, there is provided a method of detecting a TDB in acomposition, wherein the TDB comprises a target antigen-binding fragmentand a TCS-binding fragment (such as a CD3-binding fragment), the methodcomprising contacting the composition with a population of cellscomprising a) T cells comprising nucleic acid encoding a reporteroperably linked to a promoter and/or enhancer responsive to T cellactivation; and b) target cells expressing the target antigen, whereinexpression of the reporter indicates the presence of the TDB in thecomposition. In some embodiments, the reporter is a luciferase, afluorescent protein, an alkaline phosphatase, a beta lactamase, or abeta galactosidase. In some embodiments, the luciferase is a fireflyluciferase, a Renilla luciferase, or a nanoluciferase. In someembodiments, the promoter and/or enhancer responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. Insome embodiments, the promoter and/or enhancer responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1. NFκB, FOXO, STAT3, STAT5 and IRF. In someembodiments, the T cells in the population of cells are CD4⁺ T cells orCD8⁺ T cells. In some embodiments, the T cells in the population ofcells are Jurkat T cells or CELL-2 T cells. In some embodiments, theTCS-binding fragment is a CD3-binding fragment. In some embodiments, theCD3-binding fragment is a CD3ε-binding fragment. In some embodiments,the target antigen is expressed on the surface of the target cells. Insome embodiments, the target antigen is CD4, CD8, CD18, CD19, CD11a,CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGFreceptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CLL1,LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA). In some embodiments, a) the targetantigen is HER2 receptor and the target cell is a BT-474 cell, b) thetarget antigen is HER2 receptor and the target cell is a SKBR3 cell, c)the target antigen is CD20 and the target cell is a Wil2-S cell, or d)the target antigen is CD79b and the target cell is a BJAB cell. In someembodiments, the ratio of T cells to target cells in the population ofcells is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In someembodiments, the ratio of T cells to target cells in the population ofcells is about 1:4. In some embodiments, the population of cells rangesfrom about 1×10³ to about 1×10⁶. In some embodiments, the population ofcells is about 1×10⁴ to about 5×10⁴.

In some embodiments of any of the methods of detecting a TDB in acomposition described above, the population of cells is contacted with acomposition comprising the TDB at a concentration range of any of about0.01 ng/mL to about 5000 ng/mL, about 0.05 ng/mL to about 5000 ng/mL,about 0.1 ng/mL to about 5000 ng/mL, about 0.5 ng/mL to about 5000ng/mL, about 1 ng/mL to about 5000 ng/mL, about 5 ng/mL to about 5000ng/mL, about 10 ng/mL to about 5000 ng/mL, about 0.01 ng/mL to about4000 ng/mL, about 0.01 ng/mL to about 3000 ng/mL, about 0.01 ng/mL toabout 2000 ng/mL, about 0.01 ng/ml, to about 1000 ng/mL, about 0.01ng/mL to about 500 ng/mL, about 0.01 ng/mL to about 100 ng/mL, about0.01 ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about0.01 ng/mL to about 5 ng/mL, about 0.1 ng/mL to about 1000 ng/mL, about0.5 ng/mL to about 1000 ng/mL, about 1 ng/mL to about 100 ng/mL, about 1ng/mL to about 1000 ng/mL, or about 5 ng/mL to about 5000 ng/mL.

In some embodiments of any of the methods of detecting a TDB in acomposition described above, the reporter is detected after more thanabout any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10hr, 12 hr, 16 hr, 20 hr, or 24 hr after contacting the cells with thecomposition. In some embodiments, the reporter is detected between anyof about 1 hr and about 24 hr, about 1 hr and about 12 hr, about 1 hrand about 8 hr, about 1 hr and about 6 hr, about 1 hr and about 4 hr,about 1 hr and about 2 hr, about 4 hr and about 24 hr, about 4 hr andabout 12 hr, about 4 hr and about 8 hr, about 8 hr and about 24 hr,about 8 hr and about 12 hr, about 16 hr and about 24 hr, about 16 hr andabout 20 hr, or about 20 hr and about 24 hr after contacting the cellswith the composition.

In some embodiments, there is provided a method of quantifying theamount of a TDB in a composition, wherein the TDB comprises a targetantigen-binding fragment and a TCS-binding fragment (such as aCD3-binding fragment), the method comprising contacting the compositionwith a population of cells comprising a) T cells comprising nucleic acidencoding a reporter operably linked to a promoter and/or enhancerresponsive to T cell activation; and b) target cells expressing thetarget antigen, and correlating the expression of the reporter as afunction of antibody concentration with a standard curve generated bycontacting the population of T cells and target cells with differentconcentrations of a purified reference TDB. In some embodiments, thereporter is a luciferase, a fluorescent protein, an alkalinephosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the T cells in thepopulation of cells are CD4⁺ T cells or CD8⁺ T cells. In someembodiments, the T cells in the population of cells are Jurkat T cellsor CTLL-2 T cells. In some embodiments, the TCS-binding fragment is aCD3-binding fragment. In some embodiments, the CD3-binding fragment is aCD3ε-binding fragment. In some embodiments, the target antigen isexpressed on the surface of the target cells. In some embodiments, thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). Insome embodiments, a) the target antigen is HER2 receptor and the targetcell is a BT-474 cell, b) the target antigen is HER2 receptor and thetarget cell is a SKBR3 cell, c) the target antigen is CD20 and thetarget cell is a Wil2-S cell, or d) the target antigen is CD79b and thetarget cell is a BJAB cell. In some embodiments, the ratio of T cells totarget cells in the population of cells is about 1:1, about 1:2, about1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9.or about 1:10. In some embodiments, the ratio of T cells to target cellsin the population of cells is about 1:4. In some embodiments, thepopulation of cells ranges from about 1×10³ to about 1×10⁶. In someembodiments, the population of cells is about 1×10⁴ to about 5×10⁴.

In some embodiments of any of the methods of quantifying the amount of aTDB in a composition described above, the standard curve is generated bycontacting the population of cells with the purified reference TDB at aplurality of concentrations ranging from about 0.01 ng/mL to about 5000ng/mL. In some embodiments, the plurality of concentrations of purifiedreference TDB include about any one of 0.01 ng/ml, 0.1 ng /ml, 1 ng /ml,10 ng /ml, 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 500 ng/mL, 750ng/mL, 1 μg/mL, 2.5 μg/ mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 250 μg/mL, or 500 μg/mL. In some embodiments, the plurality ofconcentrations of reference TDB is about three, four, five, six, seven,eight, nine, ten or more than ten concentrations.

In some embodiments of any of the methods of quantifying the amount of aTDB in a composition described above, the reporter is detected aftermore than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9hr, 10 hr, 12 hr, 16 hr, 20 hr, or 24 hr after contacting the cells withthe composition. In some embodiments, the reporter is detected betweenany of about 1 hr and about 24 hr, about 1 hr and about 12 hr, about 1hr and about 8 hr, about 1 hr and about 6 hr, about 1 hr and about 4 hr,about 1 hr and about 2 hr, about 4 hr and about 24 hr, about 4 hr andabout 12 hr, about 4 hr and about 8 hr, about 8 hr and about 24 hr,about 8 hr and about 12 hr, about 16 hr and about 24 hr, about 16 hr andabout 20 hr, or about 20 hr and about 24 hr after contacting the cellswith the composition.

In some embodiments, there is provided a method of determining thespecificity of T cell activation mediated by a TDB, wherein the TDBcomprises a target antigen-binding fragment and a TCS-binding fragment(such as a CD3-binding fragment), the method comprising a) contacting acomposition comprising the TDB with a population of cells comprising i)T cells comprising nucleic acid encoding a reporter operably linked to apromoter and/or enhancer responsive to T cell activation; and ii) testcells that do not express the target antigen and b) contacting acomposition comprising the TDB with a population of cells comprising i)T cells comprising nucleic acid encoding a reporter operably linked to apromoter and/or enhancer responsive to T cell activation; and ii) targetcells that express the target antigen, and comparing expression of thereporter in the presence of the test cell in part a) with expression ofthe reporter in the presence of target cells in part b), wherein theratio of expression of the reporter of the test cells to the targetcells is indicative of the specificity of the TDB for the target cells.In some embodiments, the reporter is a luciferase, a fluorescentprotein, an alkaline phosphatase, a beta lactamase, or a betagalactosidase. In some embodiments, the luciferase is a fireflyluciferase, a Renilla luciferase, or a nanoluciferase. In someembodiments, the promoter and/or enhancer responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. Insome embodiments, the promoter and/or enhancer responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. In someembodiments, the T cells in the population of cells are CD4⁺ T cells orCD8⁺ T cells. In some embodiments, the T cells in the population ofcells are Jurkat T cells or CTLL-2 cells. In some embodiments, theTCS-binding fragment is a CD3-binding fragment. In some embodiments, theCD3-binding fragment is a CD3ε-binding fragment. In some embodiments,the target antigen is expressed on the surface of the target cells. Insome embodiments, the target antigen is CD4, CD8, CD18, CD19, CD11a,CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGFreceptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CLL1,LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA), in some embodiments, a) the targetantigen is HER2 receptor and the target cell is a BT-474 cell, b) thetarget antigen is HER2 receptor and the target cell is a SKBR3 cell, c)the target antigen is CD20 and the target cell is a Wil2-S cell, or d)the target antigen is CD79b and the target cell is a BJAB cell. In someembodiments, the ratio of T cells to test cells in the population ofcells of step a) and/or the ratio of T cells to target cells in thepopulation of cells of step b) is about 1:1, about 1:2, about 1:3, about1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about1:10. In some embodiments, the ratio of T cells to test cells in thepopulation of cells of step a) and/or the ratio of T cells to targetcells in the population of cells or step b) is about 1:4. In someembodiments, the population of cells of steps a) and/or b) ranges fromabout 1×10³ to about 1×10⁶. In some embodiments, the population of cellsof steps a) and/or b) ranges from about 1×10⁴ to about 5×10⁴.

In some embodiments of any of the methods of determining the specificityof T cell activation mediated by a TDB described above, the populationof T cells and test cells of step a) and the population of T cells andtarget cells of step b) are contacted with a composition comprising theTDB at a concentration range of any of about 0.01 ng mL to about 5000ng/mL, about 0.05 ng/mL to about 5000 ng/mL, about 0.1 ng/mL to about5000 ng/mL, about 0.5 ng/mL to about 5000 ng/mL, about 1 ng/mL to about5000 ng/mL, about 5 ng/mL to about 5000 ng/mL, about 10 ng/mL to about5000 ng/mL, about 0.01 ng/mL, to about 4000 ng/mL, about 0.01 ng/mL toabout 3000 ng/mL, about 0.01 ng/mL to about 2000 ng/mL, about 0.01 ng/mLto about 1000 ng/mL, about 0.01 ng/mL to about 500 ng/mL, about 0.01ng/mL to about 100 ng/mL, about 0.01 ng/mL to about 50 ng/mL, about 0.01ng/mL to about 10 ng/mL, about 0.01 ng/mL, to about 5 ng/mL, about 0.1ng/mL to about 1000 ng/mL, about 0.5 ng/mL to about 1000 ng/mL, about 1ng/mL to about 100 ng/mL, about 1 ng/mL to about 1000 ng/mL, or about 5ng/mL to about 5000 ng/mL.

In some embodiments of any of the methods of determining the specificityof T cell activation mediated by a TDB described above, the reporter isdetected after more than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 16 hr, 20 hr, or 24 hr aftercontacting the cells with the composition. In some embodiments, thereporter is detected between any of about 1 hr and about 24 hr, about 1hr and about 12 hr, about 1 hr and about 8 hr, about 1 hr and about 6hr, about 1 hr and about 4 hr, about 1 hr and about 2 hr, about 4 hr andabout 24 hr, about 4 hr and about 12 hr, about 4 hr and about 8 hr,about 8 hr and about 24 hr, about 8 hr and about 12 hr, about 16 hr andabout 24 hr, about 16 hr and about 20 hr, or about 20 hr and about 24 hrafter contacting the cells with the composition.

In some embodiments, there is provided a kit for the detection of a TDBin a composition comprising a bispecific antibody comprising a targetantigen-binding fragment and a TCS-binding fragment, wherein the kitcomprises an engineered T cell comprising a reporter operably linked toa promoter and/or enhancer that is responsive to T cell activation. Insome embodiments, the kit further comprises a reference TDB assaystandard (a purified TDB of known concentration), and/or a TDB control.In some embodiments, the kit further comprises a composition comprisingtarget cells expressing the target antigen. In some embodiments, thereporter is a luciferase, a fluorescent protein, an alkalinephosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the engineered T cellsare CD4⁺ T cells or CD8⁺ T cells. In some embodiments, the engineered Tcells are Jurkat T cells or CTLL-2 T cells. In some embodiments, theTCS-binding fragment is a CD3-binding fragment. In some embodiments, theCD3-binding fragment is a CD3ε-binding fragment. In some embodiments,the target antigen is expressed on the surface of the target cells. Insome embodiments, the target antigen is CD4, CD8, CD18, CD19, CD11a,CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGFreceptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CULL1,LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA). In some embodiments, a) the targetantigen is HER2 receptor and the target cell is a BT-474 cell, b) thetarget antigen is HER2 receptor and the target cell is a SKBR3 cell, c)the target antigen is CD20 and the target cell is a Wil2-S cell, or d)the target antigen is CD79b and the target cell is a BJAB cell. In someembodiments, the kit is used in any of the methods described above.

In some embodiments, there is provided a method of determining if apopulation of test cells expresses a target antigen, the methodcomprising a) contacting the population of test cells with a populationof T cells, wherein the T cells comprise nucleic acid encoding areporter operably linked to a promoter and/or enhancer that isresponsive to T cell activation; and b) contacting the population of Tcells and test cells with the TDB, wherein the TDB comprises a targetantigen-binding fragment and a TCS-binding fragment (such as aCD3-binding fragment), wherein expression of the reporter indicates thepresence of the target antigen expressed by the test cell. In someembodiments, the reporter is a luciferase, a fluorescent protein, analkaline phosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the population of Tcells is CD4⁺ T cells or CD8⁺ T cells. In some embodiments, thepopulation of T cells is Jurkat T cells or CTLL-2 T cells. In someembodiments, the TCS-binding fragment is a CD3-binding fragment. In someembodiments, the CD3-binding fragment is a CD3ε-binding fragment. Insome embodiments, the target antigen is expressed on the surface of thetarget cells. In some embodiments, the target antigen is CD4, CD8, CD18,CD19, CD11a, CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β(CD79b), EGF receptor, HER2 receptor, HER3 receptor, HER4 receptor,FcRH5, CLL1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αy/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA). In some embodiments, the population oftest cells are is a population of tumor cells, immune cells or vascularcells. In some embodiments, the population of test cells does notcomprise T cells. In some embodiments, the ratio of T cells to testcells is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9 or about 1:10. In some embodiments,the ratio of T cells to test cells is about 1:4. In some embodiments,the population of test cells and T cells comprises from about 1×10³ toabout 1×10⁶ cells. In some embodiments, the population of test cells andT cells comprises from about 1×10⁴ to about 5×10⁴ cells.

In some embodiments of any of the methods of determining if a populationof test cells expresses a target antigen described above, the populationof test cells and T cells is contacted with a composition comprising theTDB at a concentration range of any of about 0.01 ng/mL to about 5000ng/mL, about 0.05 ng/mL to about 5000 ng/mL, about 0.1 ng/mL to about5000 ng/m1about 0.5 ng/mL, to about 5000 ng/mL, about 1 ng/mL to about5000 ng/mL, about 5 ng/mL to about 5000 ng/mL, about 10 ng/mL to about5000 ng/mL, about 0.01 ng/mL to about 4000 ng/mL, about 0.01 ng/mL toabout 3000 ng/mL, about 0.01 ng/mL to about 2000 ng/mL, about 0.01 ng/mLto about 1000 ng/mL, about 0.01 ng/mL to about 500 ng/mL, about 0.01ng/mL to about 100 ng/mL, about 0.01 ng/mL to about 50 ng/mL, about 0.01ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.1ng/mL to about 1000 ng/mL, about 0.5 ng/mL to about 1000 ng/mL, about 1ng/mL to about 100 ng/mL, about 1 ng/mL to about 1000 ng/mL, or about 5ng/mL to about 5000 ng/mL.

In some embodiments of any of the methods of determining if a populationof test cells expresses a target antigen described above, the reporteris detected after more than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 16 hr, 20 hr, or 24 hr aftercontacting the cells with the composition. In some embodiments, thereporter is detected between any of about 1 hr and about 24 hr, about 1hr and about 12 hr, about 1 hr and about 8 hr, about 1 hr and about 6hr, about 1 hr and about 4 hr, about 1 hr and about 2 hr, about 4 hr andabout 24 hr, about 4 hr and about 12 hr, about 4 hr and about 8 hr,about 8 hr and about 24 hr, about 8 hr and about 12 hr, about 16 hr andabout 24 hr, about 16 hr and about 20 hr, or about 20 hr and about 24 hrafter contacting the cells with the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of an exemplary T cell dependentbi-specific antibody (TDB), with a first arm having binding specificityfor a target antigen and a second arm having specificity for a CD3subunit.

FIG. 2 shows a schematic representation of activation in a reporter Tcell mediated by an exemplary TDB. The reporter cell contains thefirefly luciferase reporter gene driven by NFκB response elements, whichis expressed following T cell activation mediated by bridging of thereporter T cell with a target tumor cell by the TDB.

FIG. 3A shows T cell activation by Anti-CD3 homodimer can be monitoredusing a reporter gene assay. The human Jurkat CD4⁺ T cell line wasgenetically engineered to stably express the firefly luciferase reportergene driven by various T Cell Receptor (TCR) responsive transcriptionalresponse elements (AP-1, NFAT, and NFκB), stable cell pools selected,and pools evaluated for response to treatment with 10 μg/mL of purifiedAnti-CD3 homodimer for 4 hours. Luminescence responses (luciferasereporter gene activity) were plotted, with the highest response observedfrom the Jurkat/NFκBluciferase stable pool. FIG. 3B showsJurkat/NFκBLuciferase stable clones.

FIGS. 4A and 4B show that purified anti-CD3 homodimer can activate Tcells in the presence of or absence of target cells. FIG. 4A shows acomparison of purified CD20 TDB and purified anti-CD3 homodimerpotential to activate T cells. Jurkat T cells expressing aNFκBLuciferase reporter gene are activated dose-dependently by CD20 TDBin the presence of target antigen expressing cells. CD20 TDB activatesJurkat/NFκB-fireflyLuciferase cells in the presence of the targetantigen expressing cell line. Purified CD20 TDB is 1000-fold more activethan purified anti-CD3 homodimer, in the presence of co-stimulatorytarget antigen-expressing cells. FIG. 4B shows that in the absence oftarget antigen-expressing cells (squares), CD20 TDB does not activateJurkat/NFκBLuciferase cells, but purified anti-CD3 homodimerdose-dependently induces NFκB-dependent luciferase activity (diamonds).

FIG. 5 shows T cell activation by αCD20/CD3, αHER2/CD3, and αCD79b/CD3TDBs in the presence of appropriate target cells (Wil2-S, BT-474, andBJAB cells, respectively) can be monitored using a reporter gene assaywith Jurkat/NFκB-fireflyLuciferase cells. Luminescence responses(Luciferase reporter gene activity) were plotted as a function of TDBconcentration.

FIGS. 6A and 6B show that markers of T cell activation CD69 and CD25increased in a dose-dependent manner in response to incubation with anαCD20/αCD3 TDB. FIG. 6A shows flow cytometry analysis of T cellactivation by an exemplary αCD20/αCD3 TDB, BCTC4465A, at variousconcentrations. FIG. 6B shows quantification of the flow cytometryresults.

FIG. 7 shows a comparison of the dose-response curves for T cellactivation by the αCD20/αCD3 TDB BCTC4465A, measured using either theJurkat/NFκB-fireflyLuciferase-based reporter assay or flow cytometry forpositive surface expression of CD69 and CD25.

FIG. 8 shows a schematic representation of an exemplary TDB in anELISA-based bridging binding assay, with a first arm of the TDB specificfor a HER2 epitope and a second arm of the TDB specific for a CD3εepitope. An extracellular fragment of the HER2 protein containing theHER2 epitope is coated on the surface of a plate and bridged with abiotin-labeled CD3ε peptide containing the CD3ε epitope by theanti-HER2/CD3ε TDB, and binding is detected by streptavidin-conjugatedHRP.

FIG. 9 shows that potency for T cell activation varies between twoαHER2/CD3 TDBs (αHER2/CD3 Vx and αHER2/CD3 WT) with differentCD3-binding affinities in the presence of BT-474 target cells, monitoredusing the Jurkat/NFκB-fireflyLuciferase cell-based reporter assay.Luminescence responses (luciferase reporter gene activity) were plottedas a function of TDB concentration.

FIGS. 10A and 10B show that potency for T cell activation varies betweenαHER2/CD3 TDB samples subjected to different thermal stress conditions,including no stress, 2 weeks at 40° C., and 4 weeks at 40° C., monitoredusing the Jurkat/NFκB-fireflyLuciferase cell-based reporter assay andthe ELISA-based bridging binding assay. FIG. 10A shows the relativepotencies calculated using each assay and plotted for each conditiontested. FIG. 10B shows the linear correlation between the relativepotencies for calculated using each assay.

FIG. 11 shows the potency for T cell activation of an αFcRH5/CD3 TDB inthe presence of FcRH5-expressing EJM target cells, monitored using theJurkat/NFκB-fireflyLuciferase cell-based reporter assay. Luminescenceresponses (luciferase reporter gene activity) were plotted as a functionof TDB concentration.

FIG. 12 shows potency for T cell activation of an αFcRH5/CD3 TDB usingan ELISA-based bridging assay.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for detecting T cell activation mediatedby a T cell dependent bispecific antibody (TDB) and/or determining thepotency of a TDB, wherein the TDB comprises an antigen binding fragmentthat binds to a target antigen and an antigen binding fragment thatbinds to a T cell receptor complex subunit (TCS), such as a CD3 subunit,e.g., CD3ε, expressed on a T cell, and various uses thereof, including,inter cilia, detecting a TDB in a composition, quantitating the amountof TDB in a composition, determining the specificity of a TDB, anddetermining if a population of cells expresses a target antigen.

In other aspects, the invention provides kits for detecting T cellactivation mediated by a TDB and/or determining the potency of a TDB,wherein the kit comprises an engineered T cell comprising a reporteroperably linked to a promoter and/or enhancer responsive to T cellactivation, and optionally includes the TDB, a reference TDB, a controlTDB, and/or target cells.

I. Definitions

The term “polypeptide” or “protein” are used interchangeably herein torefer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component or toxin. Also includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), as well as other modifications known in the art. The terms“polypeptide” and “protein” as used herein specifically encompassantibodies.

“Purified” polypeptide (e.g., antibody or immunoadhesin) means that thepolypeptide has been increased in purity, such that it exists in a formthat is more pure than it exists in its natural environment and/or wheninitially synthesized and/or amplified under laboratory conditions.Purity is a relative term and does not necessarily mean absolute purity.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide. In a similar manner, theterm “agonist” is used in the broadest sense and includes any moleculethat mimics a biological activity of a native polypeptide. Suitableagonist or antagonist molecules specifically include agonist orantagonist antibodies or antibody fragments, fragments or amino acidsequence variants of native polypeptides, etc. Methods for identifyingagonists or antagonists of a polypeptide may comprise contacting apolypeptide with a candidate agonist or antagonist molecule andmeasuring a detectable change in one or more biological activitiesnormally associated with the polypeptide.

A polypeptide “which binds” an antigen of interest, e.g. atumor-associated polypeptide antigen target, is one that binds theantigen with sufficient affinity such that the polypeptide is useful asa diagnostic and/or therapeutic agent in targeting a cell or tissueexpressing the antigen, and does not significantly cross-react withother polypeptides. In such embodiments, the extent of binding of thepolypeptide to a “non-target” polypeptide will be less than about 10% ofthe binding of the poly peptide to its particular target polypeptide asdetermined by fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA).

With regard to the binding of a polypeptide to a target molecule, theterm “specific binding” or “specifically binds to” or is “specific for”a particular polypeptide or an epitope on a particular polypeptidetarget means binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies including TDB)formed from at least two intact antibodies, and antibody fragments solong as they exhibit the desired biological activity. The term“immunoglobulin” (Ig) is used interchangeable with antibody herein.

Antibodies are naturally occurring immunoglobulin molecules which havevarying structures, all based upon the immunoglobulin fold. For example,IgG antibodies have two “heavy” chains and two “light” chains that aredisulphide-bonded to form a functional antibody. Each heavy and lightchain itself comprises a “constant” (C) and a “variable” (V) region. TheV regions determine the antigen binding specificity of the antibody,whilst the C regions provide structural support and function innon-antigen-specific interactions with immune effectors. The antigenbinding specificity of an antibody or antigen-binding fragment of anantibody is the ability of an antibody to specifically bind to aparticular antigen.

The antigen binding specificity of an antibody is determined by thestructural characteristics of the V region. The variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”(HVRs) that are each 9-12 amino acids long. The variable domains ofnative heavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

Each V region typically comprises three HVRs, e.g. complementaritydetermining regions (“CDRs”, each of which contains a “hypervariableloop”), and four framework regions. An antibody binding site, theminimal structural unit required to bind with substantial affinity to aparticular desired antigen, will therefore typically include the threeCDRs, and at least three, preferably four, framework regionsinterspersed there between to hold and present the CDRs in theappropriate conformation. Classical four chain antibodies have antigenbinding sites which are defined by V_(H) and V_(L) domains incooperation. Certain antibodies, such as camel and shark antibodies,lack light chains and rely on binding sites formed by heavy chains only.Single domain engineered immunoglobulins can be prepared in which thebinding sites are formed by heavy chains or light chains alone, inabsence of cooperation between V_(H) and V_(L).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” (HVR) when used herein refers to theamino acid residues of an antibody that are responsible for antigenbinding. The hypervariable region may comprise amino acid residues froma “complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (e.g. residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 52A-55(H2) and 96-101 (H3) in the V_(H) (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

As used herein, “T cell dependent bispecific” antibodies or “TDB” arebispecific antibodies designed to bind a target antigen expressed on acell, and to bind to T cells, such as by binding to a T cell receptorcomplex subunit (e.g., CD3ε) expressed on a T cell.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat.No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); one-armed antibodies, single variable domain antibodies,minibodies, single-chain antibody molecules multispecific antibodiesformed from antibody fragments (e.g., including but not limited to,Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, bi-scFv, or tandem (di,tri)-scFv);and Bi-specific T-cell engagers (BiTEs).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes).IgG1, IgG2IgG3, IgG4, IgA, and IgA2. The heavy chain constant domainsthat correspond to the different classes of antibodies are called α, δ,ε, γ, and μ, respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Plückthun in The Pharmacology of MonoclonaAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody that has polyepitopic specificity. Suchmultispecific antibodies include, but are not limited to, an antibodycomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), where the V_(H)V_(L) unit has polyepitopicspecificity, antibodies having two or more V_(L) and V_(H) domains witheach V_(H)V_(L) unit binding to a different epitope, antibodies havingtwo or more single variable domains with each single variable domainbinding to a different epitope, full length antibodies, antibodyfragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies,triabodies, tri-functional antibodies, antibody fragments that have beenlinked covalently or non-covalently. “Polyepitopic specificity” refersto the ability to specifically bind to two or more different epitopes onthe same or different target(s). “Monospecific” refers to the ability tobind only one epitope. According to one embodiment the multispecificantibody is an IgG antibody that binds to each epitope with an affinityof 5 μM to 0.001 pM, 3 μM, to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to0.001 pM, or 0.1 μM to 0.001 pM.

The expression “single domain antibodies” (sdAbs) or “single variabledomain (SVD) antibodies” generally refers to antibodies in which asingle variable domain (VH or VL) can confer antigen binding. In otherwords, the single variable domain does not need to interact with anothervariable domain in order to recognize the target antigen. Examples ofsingle domain antibodies include those derived from camelids (lamas andcamels) and cartilaginous fish (e.g., nurse sharks) and those derivedfrom recombinant methods from humans and mouse antibodies (Nature (1989)341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001)26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO03/035694; Febs Lett (1994) 339:285-290; WO00/29004; WO 02/051870).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the methods provided herein maybe made by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No, 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol,Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Nod. Acad. Sci. USA 81:6851-6855 (1984)),Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant region sequences (US Pat No.5,693,780).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence, except for FRsubstitution(s) as noted above. The humanized antibody optionally alsowill comprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin. For further details, see Joneset at., Nature 321:522-525 (1986); Riechmann et at., Nature 332:323-329(1988); and Presta, Curr. Op. Street. Blot. 2:593-596 (1992).

For the purposes herein, an “intact antibody” is one comprising heavyand light variable domains as well as an Fc region. The constant domainsmay be native sequence constant domains (e.g. human native sequenceconstant domains) or amino acid sequence variant thereof. Preferably,the intact antibody has one or more effector functions.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

A “naked antibody” is an antibody (as herein defined) that is notconjugated to a heterologous molecule, such as a cytotoxic moiety orradiolabel.

In some embodiments, antibody “effector functions” refer to thosebiological activities attributable to the Fc region (a native sequenceFc region or amino acid sequence variant Fc region) of an antibody, andvary with the antibody isotype. Examples of antibody effector functionsinclude: C1q binding and complement dependent cytotoxicity; Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Anna. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656(1998).

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. In some embodiments, the cells express atleast FcγRIII and carry out ADCC effector function. Examples of humanleukocytes that mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being preferred.

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. polypeptide anantibody)) complexed with a cognate antigen. To assess complementactivation, a CDC assay, e.g as described in Gazzano-Santoro et at., J.Immunol. Methods 202:163 (1996), may be performed.

The terms “Fe receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In some embodiments, the FcR is anative sequence human FcR. Moreover, a preferred FcR is one that bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (see Daëron, Annu. Rev. Immunol. 15:203-234 (1997)).FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al.J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to beidentified in the future, are encompassed by the term “FcR” herein. Theterm also includes the neonatal receptor, FcRn, which is responsible forthe transfer of maternal IgGs to the fetus (Guyer et al. J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Contaminants” refer to materials that are different from the desiredpolypeptide product. In some embodiments of the invention, contaminantsinclude charge variants of the polypeptide. In some embodiments of theinvention, contaminants include charge variants of an antibody orantibody fragment. In other embodiments of the invention, thecontaminant includes, without limitation: host cell materials, such asCHOP; leached Protein A; nucleic acid; a variant, fragment, aggregate orderivative of the desired polypeptide; another polypeptide; endotoxin;viral contaminant; cell culture media component, etc. In some examples,the contaminant may be a host cell protein (HCP) from, for example butnot limited to, a bacterial cell such as an E. coli cell, an insectcell, a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammaliancell, an avian cell, a fungal cell.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologouspolypeptide with the effector functions of immunoglobulin constantdomains. Structurally, the immunoadhesins comprise a fusion of an aminoacid sequence with the desired binding specificity which is other thanthe antigen recognition and binding site of an antibody (i.e., is“heterologous”), and an immunoglobulin constant domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

By “reporter molecule”, as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. The most commonly used reporter molecules in this type ofassay are either enzymes, fluorophores or radionuclide containingmolecules (i.e. radioisotopes) and chemiluminescent molecules.

As used herein “essentially the same” indicates that a value orparameter has not been altered by a significant effect. For example, anionic strength of a chromatography mobile phase at column exit isessentially the same as the initial ionic strength of the mobile phaseif the ionic strength has not changed significantly. For example, anionic strength at column exit that is within 10%, 5% or 1% of theinitial ionic strength is essentially the same as the initial ionicstrength.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that aspects and variations of the inventiondescribed herein include “consisting” and/or “consisting essentially of”aspects and variations.

ps II. Cell-Based Reporter Assays

The present invention provides cell-based assays to detect TDB-mediatedT cell activation in the presence of target cells and/or to determinethe potency of a TDB, wherein one antigen binding fragment of the TDBbinds a TCR complex subunit (such as a CD3 subunit, e.g., CD3ε) andactivates T cells and the other antigen binding fragment binds a targetantigen on the target cell. The cell-based assays are useful, interalia, for detecting a TDB in a composition, quantitating the amount ofTDB in a composition, determining the specificity of a TDB, anddetermining if a population of cells expresses a target antigen.

A. T Cell Activation

The mechanism of action of a TDB is to specifically deplete a targetantigen-expressing cell. Simultaneous binding of the TDB to a T cellreceptor (TCR) complex subunit, or TCS, (such as CD3ε) and to a targetantigen expressed on the surface of a target cell results in TCRclustering, leading to T cell activation and the cytotoxic depletion ofthe target cell. There have been many TDBs in the clinic (αCD3/αCD19,αCD3/αCD20, αCD3/αHER2; de Gast G C, et al., 1995, Cancer ImmunolImmunother. 40(6):390-396; Buhmann R, et al.,2009 Bone MarrowTransplant. 43(5):383-397; Chan J K, et al., 2006. Clin Cancer Res.12(6):1859-1867) and new versions of TDB-like bispecifics are beingevaluated to improve clinical efficacy (Chames, P. and Baty, D. 2009,MAbs 1(6):539-547; Fournier, P. and Schirrmacher, V., 2013, BioDrugs27(1):35-53). TDB bi-specifics are capable of activating both CD4⁺ andCD8⁺ T cell lineages, provided the right target-expressing cells arepresent. Activation of CD4⁺ T cells will result in the induction ofcytokine gene expression (IL-2, etc.), leading to the recruitment andactivation of other immune cells, including the expansion andproliferation of CD8⁺ T cells. CD8⁺ CTL activation results from theformation of an immunological synapse-like structure with target cellsvia TDB-mediated cellular bridging, leading to induction oftranscription of Perforin and Granzymes (A, B, C; depending on CTLsubtype), degranulization, and the localized release of Perforin andGranzymes across the ‘immunological synapse’-like interface between thetarget and effector cell, and resulting in the killing of the targetcell (Pores-Fernando, Pores-Fernando A T, Zweifach A, 2009, ImmunolRev., 231(1):160-173; Pipkin, M E, et al., 2010, Immunol Rev.,235(1):55-72). Effector cell-mediated cell killing is a relatively slowprocess requiring the stabilization of the synapse for several hours andrequires the transcriptional dependent activation of the prfl gene andgranzyme genes to ensure complete cell killing. Alternatively,CTL-mediated killing of target cells has also been shown to occur byFas-mediated apoptosis (Pardo, J, et al., 2003, Int Immunol.,15(12):1441-1450). The transcriptional regulation of the prfl, grB andFas-mediated cell killing machinery is dependent on NFAT, NFκB and STATenhancer elements located within the promoters of the genes required tomediate B cell depletion (Pipkin, M E, et al, 2010, Immunol Rev.,235(1):55-72, Pardo, J, et al., 2003, Int Immunol., 15(12):1441-1450).The strength of the interaction between the target and effector cells(immunological synapse) is dependent on other co-stimulatory moleculesfrom which signaling is also necessary to stabilize and maintain theinteraction between target and effector cell (Krogsgaard M, et al.,2003, Semin Immunol. 15(6):307-315; Pattu V, et al., 2013, FrontImmunol., 4:411; Klieger Y, et al., 2014, Eur J Immunol. 44(1):58-68;Schwartz J C, et al., 2002, Nat Immunol. 3(5):427-434). The monitoringof the transcriptional induction of target genes, through the use ofreporter gene assays, is therefore a mechanism of action(MoA)-reflective alternative assay system to observe the activation of Tcells by TDB.

T cell activation requires the spatial and kinetic reorganization ofcell surface proteins and signaling molecules at the contact site of theantigen presenting cell to form the immunological synapse. Coordinationof the activation and signaling of the TCR and co-stimulatory receptors(CD28, CD40, ICOS, etc.) and ligands regulates both the duration andsignaling that is required for T cell activation. Antigens presented onthe surface of antigen presenting cells (APCs) as MHC/peptide complexescan be recognized by TCRs on the surface of the T cell. MHC and TCRclustering initiates the recruitment and activation of signalingpathways that can lead to T cell activation, depending on the expressionof co-stimulatory and immunomodulatory receptors, which play a key rolein regulating T cell activation. Antibodies that bind to subunits of theTCR complex, such as CDR3ε (OKT3; Brown, W M, 2006, Curr Opin InvestigDrugs 7:381-388; Ferran, C et al., 1993 Exp Nephrol 1:83-89), can induceT cell activation by cross-linking TCRs and thereby mimicking theclustering of TCRs at the immunological synapse, and have been usedclinically, as well as for many years as a surrogate activators to studyTCR signaling in vitro. TCR clustering by anti-CD3 antibodies withoutco-stimulation weakly activates T cells, but still leads to T cellactivation and limited cytokine transcription and release. Anti-CD3mediated signaling has been shown to activate several transcriptionfactors, including NFAT, AP1, and NFκB (MF et al., 1995, J, Leukoc.Biol. 57:767-773; Shapiro V S et al., 1998, J. Immunol.161(12)6455-6458; Pardo, J, et al., 2003, Int Immunol.,15(12):1441-1450). Co-stimulation regulates the level and type ofcytokine release via the modulation of signaling, impactingtranscriptional regulation of cytokine expression and the nature of theT cell activation response (Shannon, M F et 471, 1995, J. Leukoc. Biol.57:767-773). The TDB clusters TCRs on the cell surface of the T cell asa result of the bridge formed between the T cell and the targetantigen-expressing cell. Transcriptional regulatory elements driving theexpression of reporter genes that may be transcriptional induced by cellactivation were tested in T cell lines to determine which events areactivated by the TDB in the presence and absence of target cells.

B. Reporter Molecules

A reporter assay is an analytical method that enables the biologicalcharacterization of a stimulus by monitoring the induction of expressionof a reporter in a cell. The stimulus leads to the induction ofintracellular signaling pathways that result in a cellular response thattypically includes modulation of gene transcription. In some examples,stimulation of cellular signaling pathways result in the modulation ofgene expression via the regulation and recruitment of transcriptionfactors to upstream non-coding regions of DNA that are required forinitiation of RNA transcription leading to protein production. Controlof gene transcription and translation in response to a stimulus isrequired to elicit the majority of biological responses such as cellularproliferation, differentiation, survival and immune responses. Thesenon-coding regions of DNA, also called enhancers, contain specificsequences that are the recognition elements for transcription factorswhich regulate the efficiency of gene transcription and thus, the amountand type of proteins generated by the cell in response to a stimulus. Ina reporter assay, an enhancer element and minimal promoter that isresponsive to a stimulus is engineered to drive the expression of areporter gene using standard molecular biology methods. The DNA is thentransfected into a cell, which contains all the machinery tospecifically respond to the stimulus, and the level of reporter genetranscription, translation, or activity is measured as a surrogatemeasure of the biological response.

In some aspects, the invention provides methods of detectingTDB-mediated T cell activation by contacting a TDB comprising aTCS-specific (such as a CD3 subunit-specific, e.g., a CD3ε-specific)antigen binding fragment and a target antigen-specific antigen bindingfragment with a population of cells comprising a) cells comprisingnucleic acid encoding a reporter operably linked to a promoter and/orenhancer responsive to T cell activation; and b) target cells presentingthe target antigen on their surface, such that expression of thereporter indicates activation of the T cells. A reporter molecule may beany molecule for which an assay can be developed to measure the amountof that molecule that is produced by the cell in response to thestimulus. For example, a reporter molecule may be a reporter proteinthat is encoded by a reporter gene that is responsive to the stimulus(e.g., T cell activation). Commonly used examples of reporter moleculesinclude, but are not limited to, luminescent proteins such asluciferase, which emit light that can be measured experimentally as aby-product of the catalysis of substrate. Luciferases are a class ofluminescent proteins that are derived from many sources and includefirefly luciferase (from the species, Photinus pyralis), Renillaluciferase from sea pansy (Renilla reniformis), click beetle luciferase(from Pyrearinus termitilluminans), marine copepod Gaussia luciferase(from Gaussia princeps), and deep sea shrimp Nano luciferase (fromOplophorus gracilirostris). Firefly luciferase catalyzes the oxygenationof luciferin to oxyluciferin, resulting in the emission of a photon oflight, while other luciferases, such as Renilla, emit light bycatalyzing the oxygenation of coelenterazine. The wavelength of lightemitted by different luciferase forms and variants can be read usingdifferent filter systems, which facilitates multiplexing. The amount ofluminescence is proportional to the amount of luciferase expressed inthe cell, and luciferase genes have been used as a sensitive reporter toquantitatively evaluate the potency of a stimulus to elicit a biologicalresponse. Reporter gene assays have been used for many years for a widerange of purposes including basic research, HTS screening, and forpotency (Brogan T, et al., 2012, Radiat Res. 177(4):508-513; Miraglia LJ, et al., 2011, Comb Chem High Throughput Screen. 14(8):648-657;Nakajima Y, and Ohmiya Y. 2010, Expert Opin Drug Discovery,5(9):835-849; Parekh B S, et al., 2012, Mabs, 4(3):310-318; Svobodova K,and Cajtham L T., 2010, Appl Microbiol Biotechnol., 88(4): 839-847).

In some embodiments, the invention provides cell-based assays to detectTDB-mediated T cell activation in T cells comprising nucleic acidencoding a reporter operably linked to a promoter and/or enhancerresponsive to T cell activation. In some embodiments, the reporterconstruct comprises a luciferase. In some embodiments, the luciferase isa firefly luciferase (e.g from the species Photinus pyralis), Renillaluciferase from sea pansy (e.g., from the species Renilla reniformis),click beetle luciferase (e.g., from the species Pyrearinustermitilluminans), marine copepod Gaussia luciferase (e.g., from thespecies Gaussia princeps), or deep sea shrimp Nano luciferase (e.g.,from the species Oplophorus gracilirostris). In some embodiments,expression of luciferase in the engineered T cell indicates theactivation of T cells by the TDB. In other aspects, the reporterconstruct encodes a β-glucuronidase (GUS); a fluorescent protein such asGreen fluorescent protein (GFP), red fluorescent protein (RFP), bluefluorescent protein (BFP), yellow fluorescent protein (YFP) or variantsthereof; a chloramphenicoal acetyltransferase (CAT); a β-galactosidase;a β-lactamase; or a secreted alkaline phosphatase (SEAP).

In some embodiments, there are provided engineered T cells comprisingnucleic acid encoding a reporter molecule (e.g., a reporter protein,such as a luciferase) operably linked to control sequences comprising apromoter and/or enhancers responsive to T cell activation. Promoterand/or enhancer sequences can be selected from among any of those knownin the art to be responsive to T cell activation. In some embodiments,the nucleic acid is stably integrated into the T cell genome.

In some embodiments, there are provided engineered T cells comprisingnucleic acid encoding a reporter molecule under the control of a minimalpromoter operably linked to one or more T cell activation responsiveenhancer elements. In some embodiments, the minimal promoter is athymidine kinase (TK) minimal promoter, a minimal promoter fromcytomegalovirus (CMV), an SV40-derived promoter, or a minimal elongationfactor 1 alpha (EF1α) promoter. In some embodiments, the minimalpromoter is a minimal TK promoter. In some embodiments, the minimalpromoter is a minimal CMV promoter. In some embodiments, the T cellactivation responsive enhancer elements are NFAT (Nuclear Factor ofActivated T cells) enhancers, AP-1 (Fos/Jun) enhancers, NFAT/AP1enhancers, NFκB enhancers, FOXO enhancers. STAT3 enhancers, STAT5enhancers or IRF enhancers. In some embodiments, the T cell activationresponsive enhancer elements are arranged as tandem repeats (such asabout any of 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). The T cellactivation responsive enhancer elements may be positioned 5′ or 3′ tothe reporter-encoding sequence. In some embodiments, the T cellactivation responsive enhancer elements are located at a site 5′ fromthe minimal promoter. In some embodiments, the T cell activationresponsive enhancer elements are NFκB enhancers. In some embodiments,the reporter molecule is a luciferase, such as firefly or Renillaluciferase. In some embodiments, the nucleic acid is stably integratedinto the T cell genome.

C. Cells

In some embodiments, there are provided methods of detectingTDB-mediated T cell activation by contacting a TDB comprising aTCS-specific (such as CD3-specific, e.g., CD3ε-specific) antigen bindingfragment and a target antigen-specific antigen binding fragment with apopulation of cells comprising a) T cells comprising nucleic acidencoding a reporter operably linked to a promoter and/or enhancerresponsive to T cell activation; and b) target cells presenting thetarget antigen on their surface, such that expression of the reporterindicates activation of the T cells In some embodiments, the T cells areCD8⁺ T cells. In yet other embodiments, the T cell is a CD4⁺/CD8⁺ Tcell. In some embodiments, the CD4⁺ and/or CD8⁺ T cells exhibitincreased release of cytokines selected from the group consisting ofIFN-γ, TNF-α, and interleukins. In some embodiments, the population of Tcells is a population of immortalized T cells (e.g., an immortalized Tcell line). In some embodiments, the population of T cells is apopulation of immortalized CD4⁺ and/or CD8⁺ cells that expressedTCR/CD3ε. In some embodiments, the T cell is a Jurkat cell. In someembodiments, the T cell is a CTLL-2 T cell.

In some embodiments, T cells of the invention comprise a cell receptor.T cell receptors exist as a complex of several proteins. The T cellreceptor itself is composed of two separate peptide chains encoded bythe independent T cell receptor alpha and beta (TCRα and TCRβ) genes.Other proteins in the complex include the CD3 proteins: CD3ε, CD3γ, CD3δand CD3ζ. The CD3 proteins are found as CD3εγ and CD3εδ heterodimers anda CD3ζ homodimer. The CD3 ζ homodimer allows the aggregation ofsignaling complexes around these proteins. In some embodiments, one armof the TDB binds a T cell receptor complex. In some embodiments, the TDBbinds CD3. In some embodiments, the TDB binds the CD3ε subunit.

In some embodiments, the invention provides compositions comprising Tcells for use in a cell-based assay to detect and/or quantitateTDB-mediated T cell activation. In some embodiments the T cells of thecomposition are CD4⁺ T cell. In some embodiments, the T cells of thecomposition are CD8⁺ T cell. In yet other embodiments, the T cells ofthe composition are CD4⁺/CD8⁺ cells. In some embodiments, the T cells ofthe composition are immortalized T cells. In some embodiments, the cellsof the composition are Jurkat cells. In some embodiments, the T cells ofthe composition are CTLL-2 T cells. In some embodiments, the T cells ofthe composition comprise a reporter construct responsive to T cellactivation. In some embodiments, the reporter construct comprises apolynucleotide encoding a luciferase. In some embodiments, theluciferase is a firefly luciferase, a Renilla luciferase, or ananoluciferase. In some embodiments, the polynucleotide encoding thereporter (e.g., luciferase) is operably linked to a T cell activationresponsive regulatory element (e.g., a cell activation responsivepromoter and/or enhancer). In some embodiments, the promoter and/orenhancer responsive to T cell activation is an NFAT promoter, an AP-1promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5promoter or an IRF promoter.

In some embodiments, the invention provides compositions of cellsengineered with a T cell activation reporter construct encoding areporter molecule operably linked to control sequences comprising apromoter and/or enhancers responsive to T cell activation. In someembodiments, the reporter molecule is a luciferase, a fluorescentprotein (e.g., a GFP, aYFP, etc.), an alkaline phosphatase, or a betagalactosidase. In some embodiments, the luciferase is a fireflyluciferase, a Renilla luciferase, or a nanoluciferase. In someembodiments, the promoter and/or enhancer responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. Insome embodiments, the enchancer responsive to T cell activationcomprises T cell responsive enhancer elements from any one or more ofNFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. In some embodiments, thecomposition of T cells comprises CD4⁺ T cells and/or CD8⁺ T cells. Insome embodiments, the T cells are Jurkat cells or CTLL-2 cells, in someembodiments, the T cells are Jurkat cells comprising a polynucleotideencoding a luciferase operably linked to an NFκB promoter.

In some embodiments, the reporter effector cells are Jurkat-Dual™ Cells(InvivoGen), Jurkat-Dual™ Cells comprise the Lucia™ secreted lucifierasegene under the control of five copies of the consensus NF-κBtranscriptional response element and three copies of the c-Rel bindingsite. The cells also comprise a secreted embryonic alkaline phosphatase(SEAP) gene under the control of an ISG54 minimal promoter and fiveinterferon-stimulated response elements.

D. TDB-Mediated T Cell Activation Assay, and Uses Thereof

In some aspects, the invention provides methods for detecting T cellactivation mediated by a TDB, wherein the TDB comprises a targetantigen-binding fragment and a TCS-binding fragment (such as aCD3-binding fragment), the method comprising contacting a compositioncomprising the TDB with a population of cells comprising a) T cellscomprising nucleic acid encoding a reporter operably linked to apromoter and/or enhancer responsive to T cell activation; and b) targetcells expressing the target antigen, wherein expression of the reporterindicates the presence of TDB-mediated T cell activation. In someembodiments, the reporter is a luciferase, a fluorescent protein, analkaline phosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the cells in thepopulation of cells are CD4⁺ T cells or CD8⁺ T cells. In someembodiments, the cells in the population of cells are Jurkat T cells orCTLL-2 T cells. In some embodiments, the TCS-binding fragment is aCD3-binding fragment. In some embodiments, the CD3-binding fragment is aCD3ε-binding fragment. In some embodiments, the target antigen isexpressed on the surface of the target cells. In some embodiments, thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). Insome embodiments, a) the target antigen is HER2 receptor and the targetcell is a BT-474 cell, b) the target antigen is HER2 receptor and thetarget cell is a SKBR3 cell, c) the target antigen is CD20 and thetarget cell is a Wil2-S cell, or d) the target antigen is CD79b and thetarget cell is a BJAB cell. In some embodiments, the ratio of T cells totarget cells in the population of cells is about 1:1, about 1:2, about1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9,or about 1:10. In some embodiments, the ratio of T cells to target cellsin the population of cells is about 1:4. In some embodiments, thepopulation of cells ranges from about 1×10³ to about 1×10⁶. In someembodiments, the population of cells is about 1×10⁴ to about 5×10⁴.

In some embodiments, the population of cells is contacted with acomposition comprising the TDB at a concentration range of any of about0.01 ng/mL to about 5000 ng/mL, about 0.05 ng/mL to about 5000 ng/mL,about 0.1 ng/mL to about 5000 ng/mL, about 0.5 ng/mL to about 5000ng/mL, about 1 ng/mL, to about 5000 ng/mL about 5 ng/mL to about 5000ng/mL, about 10 ng/mL to about 5000 ng/mL, about 0.01 ng/mL, to about4000 ng/mL, about 0.01 ng/mL to about 3000 ng/mL, about 0.01 ng/mL toabout 2000 ng/mL, about 0.01 ng/mL to about 1000 ng/mL, about 0.01 ng/mLto about 500 ng/mL, about 0.01 ng/mL to about 100 ng/mL, about 0.01ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01ng/mL, to about 5 ng/mL, about 0.1 ng/mL to about 1000 ng/mL, about 0.5ng/mL to about 1000 ng/mL, about 1 ng/mL to about 100 ng/mL, about 1ng/mL to about 1000 ng/mL, or about 5 ng/mL to about 5000 ng/mL.

In some embodiments, the reporter is detected after more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr,16 hr, 20 hr, or 24 hr after contacting the cells with the composition.In some embodiments, the reporter is detected between any of about 1 hrand about 24 hr, about 1 hr and about 12 hr, about 1 hr and about 8 hr,about 1 hr and about 6 hr, about 1 hr and about 4 hr, about 1 hr andabout 2 hr, about 4 hr and about 24 hr, about 4 hr and about 12 hr,about 4 hr and about 8 hr, about 8 hr and about 24 hr, about 8 hr andabout 12 hr, about 16 hr and about 24 hr, about 16 hr and about 20 hr,or about 20 hr and about 24 hr after contacting the cells with thecomposition.

In some aspects, the invention provides methods for detecting a TDB in acomposition, wherein the TDB comprises a target antigen-binding fragmentand a TCS-binding fragment (such as a CD3-binding fragment), the methodcomprising contacting the composition with a population of cellscomprising a) T cells comprising nucleic acid encoding a reporteroperably linked to a promoter and/or enhancer responsive to T cellactivation; and b) target cells expressing the target antigen, whereinexpression of the reporter indicates the presence of the TDB in thecomposition. In some embodiments, the reporter is a luciferase, afluorescent protein, an alkaline phosphatase, a beta lactamase, or abeta galactosidase. In some embodiments, the luciferase is a fireflyluciferase, a Renilla luciferase, or a nanoluciferase. In someembodiments, the promoter and/or enhancer responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. Insome embodiments, the promoter and/or enhancer responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. In someembodiments, the T cells in the population of cells are CD4⁺ cells orCD8⁺ T cells. In some embodiments, the T cells in the population ofcells are Jurkat T cells or CTLL-2 T cells. In some embodiments, theTCS-binding fragment is a CD3-binding fragment. In some embodiments, theCD3-binding fragment is a CD3ε-binding fragment. In some embodiments,the target antigen is expressed on the surface of the target cells. Insome embodiments, the target antigen is CD4, CD8, CD18, CD19, CD11a,CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGFreceptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CLL1,LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA). In some embodiments, a) the targetantigen is HER2 receptor and the target cell is a BT-474 cell, b) thetarget antigen is HER2 receptor and the target cell is a SKBR3 cell, c)the target antigen is CD20 and the target cell is a Wil2-S cell, or d)the target antigen is CD79b and the target cell is a BJAB cell. In someembodiments, the ratio of T cells to target cells in the population ofcells is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In someembodiments, the ratio of T cells to target cells in the population ofcells is about 1:4. In some embodiments, the population of cells r sfrom about 1×10³ to about 1×10⁶ some embodiments, the population ofcells is about 1×10⁴ to about 5×10⁴.

In some embodiments, the population of cells is contacted with acomposition comprising the TDB at a concentration range of any of about0.01 ng/mL to about 5000 ng/mL, about 0.05 ng/mL to about 5000 ng/mL,about 0.1 ng/mL to about 5000 ng/mL, about 0.5 ng/mL to about 5000ng/mL, about 1 ng/mL to about 5000 ng/mL, about 5 ng/mL to about 5000ng/mL, about 10 ng/mL to about 5000 ng/mL, about 0.01 ng/mL to about4000 ng/mL, about 0.01 ng/mL to about 3000 ng/mL, about 0.01 ng/mL toabout 2000 ng/mL, about 0.01 ng/mL to about 1000 ng/mL, about 0.01 ng/mLto about 500 ng/mL, about 0.01 ng/mL to about 100 ng/mL, about 0.01ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01ng/mL, to about 5 ng/mL, about 0.1 ng/mL to about 1000 ng/mL, about 0.5ng/mL to about 1000 ng/mL, about 1 ng/mL to about 100 ng/mL, about 1ng/mL to about 1000 ng/mL, or about 5 ng/mL to about 5000 ng/mL.

In some embodiments, the reporter is detected after more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr,16 hr, 20 hr, or 24 hr after contacting the cells with the composition.In some embodiments, the reporter is detected between any of about 1 hrand about 24 hr, about 1 hr and about 12 hr, about 1 hr and about 8 hr,about 1 hr and about 6 hr, about 1 hr and about 4 hr, about 1 hr andabout 2 hr, about 4 hr and about 24 hr, about 4 hr and about 12 hr,about 4 hr and about 8 hr, about 8 hr and about 24 hr, about 8 hr andabout 12 hr, about 16 hr and about 24 hr, about 16 hr and about 20 hr,or about 20 hr and about 24 hr after contacting the cells with thecomposition.

In some aspects, the invention provides methods for quantifying theamount of a TDB in a composition, wherein the TDB comprises a targetantigen-binding fragment and a TCS-binding fragment (such as aCD3-binding fragment), the method comprising contacting the compositionwith a population of cells comprising a) T cells comprising nucleic acidencoding a reporter operably linked to a promoter and/or enhancerresponsive to T cell activation; and b) target cells expressing thetarget antigen, and correlating the expression of the reporter as afunction of antibody concentration with a standard curve generated bycontacting the population of T cells and target cells with differentconcentrations of a purified reference TDB. In some embodiments, thereporter is a luciferase, a fluorescent protein, an alkalinephosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the T cells in thepopulation of cells are CD4⁺ T cells or CD8⁺ T cells. In someembodiments, the T cells in the population of cells are Jurkat T cellsor CTLL-2 T cells. In some embodiments, the TCS-binding fragment is aCD3-binding fragment. In some embodiments, the CD3-binding fragment is aCD3ε-binding fragment. In some embodiments, the target antigen isexpressed on the surface of the target cells. In some embodiments, thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). Insome embodiments, a) the target antigen is HER2 receptor and the targetcell is a BT-474 cell, b) the target antigen is HER2 receptor and thetarget cell is a SKBR3 cell, c) the target antigen is CD20 and thetarget cell is a Wil2-S cell, or d) the target antigen is CD79b and thetarget cell is a BJAB cell. In some embodiments, the ratio of T cells totarget cells in the population of cells is about 1:1, about 1:2, about1:3, about 1:4, about 1:5, about 1:6, about 1:7_(;) about 1:8, about1:9, or about 1:10. In some embodiments, the ratio of T cells to targetcells in the population of cells is about 1:4. In some embodiments, thepopulation of cells ranges from about 1×10³ to about 1×10⁶. In someembodiments, the population of cells is about 1×10⁴ to about 5×10⁴.

In some embodiments, the standard curve is generated by contacting thepopulation of cells with the purified reference TDB at a plurality ofconcentrations ranging from about 0.01 ng/mL to about 5000 ng/mL. Insome embodiments, the plurality of concentrations of purified referenceTDB include about any one of 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml,100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 2.5μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, or500 μg/mL. In some embodiments, the plurality of concentrations ofreference TDB is about three, four, five, six, seven, eight, nine, tenor more than ten concentrations.

In some embodiments, the reporter is detected after more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr,16 hr, 20 hr, or 24 hr after contacting the cells with the composition.In some embodiments, the reporter is detected between any of about 1 hrand about 24 hr, about 1 hr and about 12 hr, about 1 hr and about 8 hr,about 1 hr and about 6 hr, about 1 hr and about 4 hr, about 1 hr andabout 2 hr, about 4 hr and about 24 hr, about 4 hr and about 12 hr,about 4 hr and about 8 hr, about 8 hr and about 24 hr, about 8 hr andabout 12 hr, about 16 hr and about 24 hr, about 16 hr and about 20 hr,or about 20 hr and about 24 hr after contacting the cells with thecomposition.

In some aspects, the invention provides methods for determining thepotency of T cell activation mediated by a TDB, wherein the TDBcomprises a target antigen-binding fragment and a TCS-binding fragment(such as a CD3-binding fragment), the method comprising contacting acomposition comprising the TDB with a population of cells comprising a)T cells comprising nucleic acid encoding a reporter operably linked to apromoter and/or enhancer responsive to T cell activation; and b) targetcells expressing the target antigen, and correlating the expression ofthe reporter as a function of antibody concentration with a standardcurve generated by contacting the population of cells with differentconcentrations of a reference TDB, thereby obtaining a relative measureof the potency of T cell activated mediated by the TDB. In someembodiments, the reporter is a luciferase, a fluorescent protein, analkaline phosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the T cells in thepopulation of cells are CD4⁺ T cells or CD8⁺ T cells. In someembodiments, the T cells in the population of cells are Jurkat T cellsor CTLL-2 T cells. In some embodiments, the TCS-binding fragment is aCD3-binding fragment. In some embodiments, the CD3-binding fragment is aCD3ε-binding fragment. In some embodiments, the target antigen isexpressed on the surface of the target cells. In some embodiments, thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α, (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). Insome embodiments, a) the target antigen is HER2 receptor and the targetcell is a BT-474 cell, b) the target antigen is HER2 receptor and thetarget cell is a SKBR3 cell, c) the target antigen is CD20 and thetarget cell is a Wil2-S cell, or d) the target antigen is CD79b and thetarget cell is a BJAB cell. In some embodiments, the ratio of T cells totarget cells in the population of cells is about 1:1. about 1:2, about1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9,or about 1:10. In some embodiments, the ratio of T cells to target cellsin the population of cells is about 1:4. In some embodiments, thepopulation of cells ranges from about 1×10³ to about 1×10⁶. In someembodiments, the population of cells is about 1×10⁴ to about 5×10⁴.

In some embodiments, the population of cells is contacted with acomposition comprising the TDB at a concentration range of any of about0.01 ng/mL to about 5000 ng/mL, about 0.05 ng/mL to about 5000 ng/mL,about 0.1 ng/mL to about 5000 ng/mL, about 0.5 ng/mL to about 5000ng/mL, about 1 ng/mL to about 5000 ng/mL, about 5 ng/mL to about 5000ng/mL, about 10 ng/mL to about 5000 ng/mL, about 0.01 ng/mL to about4000 ng/mL, about 0.01 ng/mL to about 3000 ng/mL, about 0.01 ng/mL toabout 2000 ng/mL, about 0.01 ng/mL to about 1000 ng/mL, about 0.01 ng/mLto about 500 ng/mL, about 0.01 ng/mL, to about 100 ng/mL, about 0.01ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01ng/mL to about 5 ng/mL, about 0.1 ng/mL to about 1000 ng/mL, about 0.5ng/mL to about 1000 ng,/mL, about 1 ng/mL to about 100 ng/mL, about 1ng/mL to about 1000 ng/mL, or about 5 ng/mL to about 5000 ng/mL.

In some embodiments, the standard curve is generated by contacting thepopulation of cells with the purified reference TDB at a plurality ofconcentrations ranging from about 0.01 ng/mL to about 5000 ng/mL. Insome embodiments, the plurality of concentrations of purified referenceTDB include about any one of 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml,100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 1μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250μg/mL, or 500 μg/mL. In some embodiments, the plurality ofconcentrations of reference TDB is about three, four, five, six, seven,eight, nine, ten or more than ten concentrations.

In some embodiments, the reporter is detected after more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr,16 hr, 20 hr, or 24 hr after contacting the cells with the composition.In some embodiments, the reporter is detected between any of about 1 hrand about 24 hr, about 1 hr and about 12 hr, about 1 hr and about 8 hr,about 1 hr and about 6 hr, about 1 hr and about 4 hr, about 1 hr andabout 2 hr, about 4 hr and about 24 hr, about 4 hr and about 12 hr,about 4 hr and about 8 hr, about 8 hr and about 24 hr, about 8 hr andabout 12 hr, about 16 hr and about 24 hr, about 16 hr and about 20 hr,or about 20 hr and about 24 hr after contacting the cells with thecomposition.

In some aspects, the invention provides methods for determining thespecificity of T cell activation mediated by a TDB, wherein the TDBcomprises a target antigen-binding fragment and a TCS-binding fragment(such as a CD3-binding fragment), the method comprising a) contacting acomposition comprising the TDB with a population of cells comprising i)T cells comprising nucleic acid encoding a reporter operably linked to apromoter and/or enhancer responsive to T cell activation; and ii) testcells that do not express the target antigen; and b) contacting acomposition comprising the TDB with a population of cells comprising i)T cells comprising nucleic acid encoding a reporter operably linked to apromoter and/or enhancer responsive to T cell activation; and ii) targetcells that express the target antigen, and comparing expression of thereporter in the presence of the test cell in part a) with expression ofthe reporter in the presence of target cells in part b), wherein theratio of expression of the reporter of the test cells to the targetcells is indicative of the specificity of the TDB for the target cells.In some embodiments, the reporter is a luciferase, a fluorescentprotein, an alkaline phosphatase, a beta lactamase, or a betagalactosidase. In some embodiments, the luciferase is a fireflyluciferase, a Renilla luciferase, or a nanoluciferase. In someembodiments, the promoter and/or enhancer responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. Insome embodiments, the promoter and/or enhancer responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. In someembodiments, the T cells in the population of cells are CD4⁺ T cells orCD8⁺ T cells. In some embodiments, the T cells in the population ofcells are Jurkat T cells or CTLL-2 cells. In some embodiments, theTCS-binding fragment is a CD3-binding fragment. In some embodiments, theCD3-binding fragment is a CD3ε-binding fragment. In some embodiments,the target antigen is expressed on the surface of the target cells. Insome embodiments, the target antigen is CD4, CD8, CD18, CD19, CD11a,CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGFreceptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CLL1,LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, TCAM, αv/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA). In some embodiments, a) the targetantigen is HER2 receptor and the target cell is a BT-474 cell, b) thetarget antigen is HER2 receptor and the target cell is a SKBR3 cell, c)the target antigen is CD20 and the target cell is a Wil2-S cell, or d)the target antigen is CD79b and the target cell is a BJAB cell. In someembodiments, the ratio of T cells to test cells in the population ofcells of step a) and/or the ratio of T cells to target cells in thepopulation of cells of step b) is about 1:1, about 1:2, about 1:3, about1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about1:10. In some embodiments, the ratio of T cells to test cells in thepopulation of cells of step a) and/or the ratio of T cells to targetcells in the population of cells or step b) is about 1:4. In someembodiments, the population of cells of steps a) and/or b) ranges fromabout 1×10³ to about 1×10⁶. In some embodiments, the population of cellsof steps a) and/or b) ranges from about 1×10⁴ to about 5×10⁴.

In some embodiments, the population of T cells and test cells of step a)and the population of T cells and target cells of step b) are contactedwith a composition comprising the TDB at a concentration range of any ofabout 0.01 ng/mL, to about 5000 ng/mL, about 0.05 ng/mL to about 5000ng/mL, about 0.1 ng/mL to about 5000 ng/mL, about 0.5 ng/mL to about5000 ng/mL, about 1 ng/mL to about 5000 ng/mL, about 5 ng/mL to about5000 ng/mL, about 10 ng/mL to about 5000 ng/mL, about 0.01 ng/mL toabout 4000 ng/mL, about 0.01 ng/mL to about 3000 ng/mL, about 0.01 ng/mLto about 2000 ng/mL, about 0.01 ng/mL to about 1000 ng/mL, about 0.01ng/mL to about 500 ng/mL, about 0.01 ng/mL to about 100 ng/mL, about0.01 ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about0.01 ng/mL to about 5 ng/mL, about 0.1 ng/mL to about 1000 ng/mL, about0.5 ng/mL to about 1000 ng/mL, about 1 ng/mL to about 100 ng/mL, about 1ng/mL to about 1000 ng/mL, or about 5 ng/mL to about 5000 ng/mL.

In some embodiments, the reporter is detected after more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr,16 hr, 20 hr, or 24 hr after contacting the cells with the composition.In some embodiments, the reporter is detected between any of about 1 hrand about 24 hr, about 1 hr and about 12 hr, about 1 hr and about 8 hr,about 1 hr and about 6 hr, about 1 hr and about 4 hr, about 1 hr andabout 2 hr, about 4 hr and about 24 hr, about 4 hr and about 12 hr,about 4 hr and about 8 hr, about 8 hr and about 24 hr, about 8 hr andabout 12 hr, about 16 hr and about 24 hr, about 16 hr and about 20 hr,or about 20 hr and about 24 hr after contacting the cells with thecomposition.

In some aspects, the invention provides methods for determining if apopulation of test cells expresses a target antigen, the methodcomprising a) contacting the population of test cells with a populationof T cells, wherein the T cells comprise nucleic acid encoding areporter operably linked to a promoter and/or enhancer that isresponsive to T cell activation; and b) contacting the population of Tcells and test cells with the TDB, wherein the TDB comprises a targetantigen-binding fragment and a TCS-binding fragment (such as aCD3-binding fragment), wherein expression of the reporter indicates thepresence of the target antigen expressed by the test cell. In someembodiments, the reporter is a luciferase, a fluorescent protein, analkaline phosphatase, a beta lactamase, or a beta galactosidase. In someembodiments, the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase. In some embodiments, the promoterand/or enhancer responsive to T cell activation is an NFAT promoter, anAP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, aSTAT5 promoter or an IRF promoter. In some embodiments, the promoterand/or enhancer responsive to T cell activation comprises T cellactivation responsive elements from any one or more of NFAT, AP-1, NFκB,FOXO, STAT3, STAT5 and IRF. In some embodiments, the population of Tcells is a population of CD4⁺ T cells or CD8⁺ T cells. In someembodiments, the population of T cells is a population of Jurkat T cellsor CTLL-2 T cells. In some embodiments, the TCS-binding fragment is aCD3-binding fragment. In some embodiments, the CD3-binding fragment is aCD3ε-binding fragment. In some embodiments, the target antigen isexpressed on the surface of the target cells, in some embodiments, thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). Insome embodiments, the population of test cells is a population of tumorcells, immune cells or vascular cells. In some embodiments, thepopulation of test cells does not comprise T cells. In some embodiments,the ratio of T cells to test cells is about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 orabout 1:10. In some embodiments, the ratio of T cells to test cells isabout 1:4. In some embodiments, the population of test cells and T cellscomprises from about 1×10³ to about 1×10⁶ cells, in some embodiments,the population of test cells and T cells comprises from about 1×10⁴ toabout 5×10⁴ cells.

In some embodiments, the population of test cells and T cells iscontacted with a composition comprising the TDB at a concentration rangeof any of about 0.01 ng/mL to about 5000 ng/mL, about 0.05 ng/mL toabout 5000 ng/mL, about 0.1 ng/mL to about 5000 ng/mL, about 0.5 ng/mLto about 5000 ng/mL, about 1 ng/mL to about 5000 ng/mL, about 5 ng/mL toabout 5000 ng/mL, about 10 ng/mL to about 5000 ng/mL, about 0.01 ng/mLto about 4000 ng/mL, about 0.01 ng/mL to about 3000 ng/mL, about 0.01ng/mL to about 2000 ng/mL, about 0.01 ng/mL to about 1000 ng/mL, about0.01 ng/mL to about 500 ng/mL, about 0.01 ng/mL to about 100 ng/mL,about 0.01 ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL,about 0.01 ng/mL to about 5 ng/mL, about 0.1 ng/mL to about 1000 ng/mL,about 0.5 ng/mL to about 1000 ng/mL, about 1 ng/mL to about 100 ng/mL,about 1 ng/mL to about 1000 ng/mL, or about 5 ng/mL to about 5000 ng/mL.

In some embodiments, the reporter is detected after more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr,16 hr, 20 hr, or 24 hr after contacting the cells with the composition.In some embodiments, the reporter is detected between any of about 1 hrand about 24 hr, about 1 hr and about 12 hr, about 1 hr and about 8 hr,about 1 hr and about 6 hr, about 1 hr and about 4 hr, about 1 hr andabout 2 hr, about 4 hr and about 24 hr, about 4 hr and about 12 hr,about 4 hr and about 8 hr, about 8 hr and about 24 hr, about 8 hr andabout 12 hr, about 16 hr and about 24 hr, about 16 hr and about 20 hr,or about 20 hr and about 24 hr after contacting the cells with thecomposition.

E. Assay Development

The following is an exemplary but non-limiting method of developing acell-based assay to detect TDB-mediated T cell activity.

DNA constructs: Lentivirus is used to generate the stable reporter Tcell lines used to evaluate the potency of the TDB bi-specific antibody.Lentiviral vectors are constructed that express the reporter genefirefly luciferase, Renilla luciferase, or Nanoluciferase under thecontrol of a minimal TK promoter regulated by DNA recognition elementsfor NFAT (Nuclear Factor of Activated T cells), AP-1 (Fos/Jun), MAT/AP1,NFκB, FOXO, STAT3/5, or IRF. The lentiviral expression cassettes usedfor the generation of the stable reporter cell lines may be thirdgeneration self-inactivating bi-cistronic vectors that express variousantibiotic selection markers under the control of constitutivepromoters/enhancers (EF1alpha or SV40) to enable the generation ofstable cell lines. The reporter lentiviral vectors used are modifiedfrom the pCDH.MCS.EF1a. Puro commercially available vector (SBIbiosciences; Cat No. CD510B-1). Promoter modifications include theremoval of the CMV minimal promoter and substitution with variousenhancer elements (NFAT, NFκB, etc.), addition of a minimal core RNApolymerase promoter (TATA box) from pRK5.CMV. Luciferase (Osaka, G etal., 1996 J Pharm Sci. 1996, 85:612-618), and substitution of differentselection cassettes from internal DNAs (Neomycin resistance gene frompRK5.tk.neo, Hygromycin resistance gene from pRK5.tk.hygro, and theblasticidin resistance gene from pRK5.tk.blastocidin). Impact of theconstitutive promoters used for selection on the activation of theenhancer elements is minimal due to the incorporation of a non-codingstretch of DNA designed to minimize promoter/enhancer cross-talk.Firefly Luciferase from pRK5.CMV.Luciferase (Osaka, 1996) is cloned intothe HindIII-NotI site of the modified lentiviral parent vector. Otherluminescent proteins including Renilla Luciferase and NanoLuciferase mayalso be subcloned into the HindIII-NotI site. Lentiviral packagingconstructs (pCMV.HIVdelta, pCMC.VSV-G, and pCMV.Rev) used to generateviral stocks from transient transfection of 293s (293 suspension adaptedcell line) cells may be obtained (pCMV.VSV-G) or generated(pCMV.HIVdelta, pCMV.REV). HIV strain MN (Nakamura, G R et al., 1993, J.Virol. 67(10):6179-6191) may be used to generate the pCMV.HIVdeltapackaging vector and contains an internal EcoRI partial digest deletionto inactivate by deletion the HIV viral envelope and modifications tothe 5′ and 3′LTRs for safety purposes. HIV Rev is cloned frompCMV.HIVdelta transfected 293s cell RNA by RT-PCR and introduced intothe ClaI-Xho site of pRK5.tk.neo. The use of VSV-G to pseudotype thelentiviral reporters (substituting VSV-G for HIV env) enables theinfection of any cell type. Lentiviral expression plasmids and packagingconstructs are amplified in Stbl2 competent cells (Life Technologies,Cat. No. 10268-019) and DNA purified using Qiagen Maxi Prep kit (Cat.No. 12662). All DNA constructs are confirmed by DNA sequencing.

Reporter gene assay cell line development: Jurkat CD4+ T cell line(DSMZ, Cat. No. ACC 282) and CTLL-2 CD8⁺ cell line (Life Technologies,Cat. No. K1653) are used to evaluate the feasibility of a reporter geneassay to monitor the activation of T cells by the TDB. Lentiviralvectors are constructed that express the reporter gene fireflyluciferase, Renilla luciferase, or Nanoluciferase under the control of aminimal TK promoter regulated by DNA recognition elements for NFAT(Nuclear Factor of Activated T cells), AP-1 (Fos/Jun), NFAT/AP1, NFκB,FOXO, STAT3,5, and IRF. Reporter gene viral stocks are generated bytransient transfection of 293s cells and pseudotyped with VSV-G,concentrated, and titered using standard methods (Naldini, L., et al,1996 Science, 272:263-267). The Jurkat CTLL-2 cells are infected withthe lentiviral reporter viral stock at an MOI of 10 by spinoculation andafter 3 days infected cells are selected for antibiotic resistance.After 2 weeks, stable pools are generated and evaluated for the responseto purified TDB. A qPCR method that evaluates copy number andintegration is used to demonstrate that all stable pools are stablyinfected with the reporter constructs. On the basis of theseexperiments, limiting dilution of Jurkat/NFκB-luciferase andJurkat/NFAT-Luciferase are set up to enable single cell cloning andgeneration of single stable reporter cell lines.

Development and evaluation of the T cell activation assay: To quantitatethe potency of TDB-mediated T cell activation, the amount of luciferaseactivity observed for a plurality of dilutions of a TDB test sampleincubated with a population of Jurkat/NFκB-fireflyLuciferase effectorcells and target cells is compared to the luciferase activity observedfor a reference TDB. The relative potency of the test TDB samples isdetermined from the standard curve generated by using the reference TDB.

III. Non Cell-Based Reporter Assays

The present invention in some aspects provides non cell-based assays todetect simultaneous TDB binding of a target antigen and a TCS (such as aCD3 subunit), wherein one antigen binding fragment of the TDB binds thetarget antigen and the other antigen binding fragment binds the TCS.Simultaneous binding of the TDB to a T cell receptor (TCR) complexsubunit (such as a CD3 subunit, e.g., CD3ε) and to a target antigenexpressed on the surface of a target cell results in TCR clustering,leading to T cell activation and the cytotoxic depletion of the targetcell. These non cell-based assays serve as a surrogate measure of T cellactivation.

In some embodiments, the invention provides methods of detectingsimultaneous binding of a TDB to a target antigen and a TCS (such as aCD3 subunit), wherein one antigen binding fragment of the TDB hinds afirst epitope on the target antigen and the other antigen bindingfragment binds a second epitope on the TCS, the method comprisingperforming an ELISA-based bridging binding assay using immobilizedtarget antigen, or a fragment thereof comprising the first epitope, anda conjugate of biotin and the TCS, or a fragment thereof comprising thesecond epitope. In some embodiments, the first epitope is localized toan extracellular portion of the target antigen and/or the second epitopeis localized to an extracellular portion of the TCS.

In some embodiments, the invention provides methods of detectingsimultaneous binding of a TDB to a target antigen and a TCS (such as aCD3 subunit), wherein one antigen binding fragment of the TDB binds afirst epitope on the target antigen and the other antigen bindingfragment binds a second epitope on the TCS, the method comprising a)immobilizing the target antigen, or a fragment thereof comprising thefirst epitope, to a solid phase; b) incubating the TDB with the targetantigen, or fragment thereof comprising the first epitope, immobilizedto the solid phase; c) incubating the TDB with a conjugate of a reportermolecule and the TCS, or a fragment thereof comprising the secondepitope (biotin-TCS conjugate); d) optionally incubating thereporter-TCS conjugate with an accessory molecule needed to detect thereporter molecule; e) removing molecules unbound to the solid phase(such as by washing); and f) detecting the reporter molecule bound tothe solid phase using a detection agent, thereby detecting simultaneousbinding of the TDB to the target antigen and the TCS. In someembodiments, the invention provides methods of detecting simultaneousbinding of a TDB to a target antigen and a TCS (such as a CD3 subunit),wherein one antigen binding fragment of the TDB binds a first epitope onthe target antigen and the other antigen binding fragment binds a secondepitope on the TCS, the method comprising a) immobilizing the targetantigen, or a fragment thereof comprising the first epitope, to a solidphase; b) incubating the TDB with the target antigen, or fragmentthereof comprising the first epitope, immobilized to the solid phase; c)incubating the TDB with a conjugate of biotin and the TCS, or a fragmentthereof comprising the second epitope (biotin-TCS conjugate); d)incubating the biotin-TCS conjugate with a streptavidin-HRP conjugate;e) removing molecules unbound to the solid phase (such as by washing);and f) detecting HRP bound to the solid phase using a detection agent,thereby detecting simultaneous binding of the TDB to the target antigenand the TCS. Washing steps may be included between the incubation stepsto remove molecules unbound to the solid phase. In some embodiments, theincubation steps are independently carried out for about any of 1 hr, 2hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 16 hr, 20hr, 24 hr, or more, including any ranges between these values. In someembodiments, the TCS is a CD3 subunit. In some embodiments, the CD3subunit is CD3ε. In some embodiments, the target antigen is expressed onthe surface of a cell. In some embodiments, the target antigen is CD4,CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a),CD79β (CD79b), EGF receptor, HER2 receptor, HER3 receptor, HER4receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3integrin, VEGF, flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;CTLA-4; protein C, BR3, c-met, tissue factor, β7, Tenb2, STEAP, ortransmembrane tumor-associated antigens (TAA). In some embodiments, thefirst epitope is localized to an extracellular portion of the targetantigen and/or the second epitope is localized to an extracellularportion of the TCS.

In some embodiments, the TDB included in an incubation step is at aconcentration range of any of about 0.01 ng/mL to about 100 μg/mL, about005 ng/mL to about 100 μg/mL, about 0.1 ng/mL, to about 100 μg/mL, about0.5 ng/mL to about 100 μg/mL, about 1 ng/mL, to about 100 μg/mL, about 5ng/mL to about 100 μg/mL, about 10 ng/mL to about 100 μg/mL, about 0.01ng/mL to about 50 μg/mL, about 0.01 ng/mL to about 10 μg/mL, about 0.01to about 1 μg/mL, about 0.01 ng/mL to about 100 ng/mL, about 0.01 ng/mL,to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mLto about 0.1 ng/mL, about 0.01 ng/mL to about 0.05 ng/mL, about 0.01ng/mL to about 0.05 ng/mL, about 0.1 ng/mL to about 1 μg/mL, about 0.5ng/mL to about 1 μg/mL to about 1 ng/mL to about 100 ng/mL, about 1ng/mL, to about 1 μg/mL, or about 5 ng/mL, to about 5 μg/mL.

In some embodiments, the invention provides methods of quantifyingsimultaneous binding of a TDB to a target antigen and a TCS (such as aCD3 subunit), wherein one antigen binding fragment of the TDB binds afirst epitope on the target antigen and the other antigen bindingfragment binds a second epitope on the TCS, the method comprising a)immobilizing the target antigen, or a fragment thereof comprising thefirst epitope, to a solid phase; b) incubating the TDB with the targetantigen, or fragment thereof comprising the first epitope, immobilizedto the solid phase; incubating the TDB with a conjugate of a reporterand the TCS, or a fragment thereof comprising the second epitope (e.g.,biotin-TCS conjugate); d) optionally incubating the reporter-TCSconjugate with an accessory reporter molecule; e) removing moleculesunbound to the solid phase (such as by washing); f) detecting reporterbound to the solid phase using a detection agent; and g) determining therelative potency of the TDB by comparing the signal intensity of thedetection agent to a standard generated using a reference TDB. In someembodiments, the invention provides methods of quantifying simultaneousbinding of a TDB to a target antigen and a TCS (such as a CD3 subunit),wherein one antigen binding fragment of the TDB binds a first epitope onthe target antigen and the other antigen binding fragment binds a secondepitope on the TCS, the method comprising a) immobilizing the targetantigen, or a fragment thereof comprising the first epitope, to a solidphase; b) incubating the TDB with the target antigen, or fragmentthereof comprising the first epitope, immobilized to the solid phase; c)incubating the TDB with a conjugate of biotin and the TCS, or a fragmentthereof comprising the second epitope (biotin-TCS conjugate); d)incubating the biotin-TCS conjugate with a streptavidin-HRP conjugate;e) removing molecules unbound to the solid phase (such as by washing);f) detecting HRP bound to the solid phase using a detection agent; andg) determining the relative potency of the TDB by comparing the signalintensity of the detection agent to a standard generated using areference TDB. In some embodiments, comparing the signal intensitycomprises generating a dose-response curve for each of the TDB and thereference TDB, and determining the ratio between the EC₅₀ values derivedfrom the curves. Washing steps may be included between the incubationsteps to remove molecules unbound to the solid phase. In someembodiments, the incubation steps are independently carried out forabout any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10hr, 12 hr, 16 hr, 20 hr, 24 hr, or more, including any ranges betweenthese values. In some embodiments, the TCS is a CD3 subunit. In someembodiments, the CD3 subunit is CD3ε. In some embodiments, the targetantigen is expressed on the surface of a cell. In some embodiments, thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79 (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor. HER3receptor, HER4 receptor, FcRH5, CLL1, Mac1, p150, 95, VLA-4, ICAM-1,VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB) receptor;mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, β7, Tenb2,STEAP, or transmembrane tumor-associated antigens (TAA). In someembodiments, the first epitope is localized to an extracellular portionof the target antigen and/or the second epitope is localized to anextracellular portion of the TCS.

In some embodiments, the TDB included in an incubation step is at aconcentration range of any of about 0.01 ng/mL to about 100 μg/mL, about0.05 ng/mL to about 100 μg/mL, about 0.1 ng/mL to about 100 μg/mL, about0.5 ng/mL to about 100 μg/mL, about 1 ng/mL, to about 100 μg/mL about 5ng/mL to about 100 μg/mL, about 10 ng/mL to about 100 μg/mL, about 0.01ng/mL, to about 50 μg/mL, about 0.01 ng/mL to about 10 μg/mL, about 0.01ng/mL to about 1 μg/mL, about 0.01 ng/mL to about 100 ng/mL, about 0.01ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01ng/mL to about 0.1 ng/mL, about 0.01 ng/mL to about 0.05 ng/mL, about0.01 ng/mL to about 0.05 ng/mL, about 0.1 ng/mL to about 1 μg/mL, about0.5 ng/mL to about 1 ng/mL about 1 ng/mL to about 100 ng/mL, about 1ng/mL to about 1 μg/mL, or about 5 ng/mL to about 5 μg/mL.

In some embodiments, the standard curve from the reference TDB isgenerated by incubating the reference TDB at a plurality ofconcentrations ranging from about any one of 0.01 ng/mL to 100 μg/mL. Insome embodiments, the plurality of concentrations of reference TDBinclude about any one of 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100ng/mL, 250 ng/mL, 500 ng/mL, 1 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 25μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, or 500 μg/mL. In someembodiments, the plurality of concentrations of reference TDB is aboutthree, four, five, six, seven, eight, nine, ten or more than tenconcentrations.

IV. Kits

In some aspects of the invention, a kit or article of manufacture isprovided for use in various methods involving a TDB comprising a targetantigen-binding fragment and a TCS-binding fragment (such as aCD3-binding fragment), comprising a container which holds a compositioncomprising engineered T cells comprising nucleic acid encoding areporter operably linked to a promoter and/or enhancers that areresponsive to T cell activation as described herein, and optionallyprovides instructions for its use. In some embodiments, the kit furthercomprises a container which holds a reference TDB assay standard (apurified TDB of known concentration), and/or a container which holds aTDB control. In some embodiments, the kit further comprises a containerwhich holds a composition comprising target cells expressing the targetantigen. In some embodiments, the reporter is a luciferase, afluorescent protein, an alkaline phosphatase, a beta lactamase, or abeta galactosidase. In some embodiments, the luciferase is a fireflyluciferase, a Renilla luciferase, or a nanoluciferase. In someembodiments, the promoter and/or enhancer responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. Insome embodiments, the promoter and/or enhancer responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. In someembodiments, the engineered T cells are CD4⁺ T cells or CD8⁺ T cells. Insome embodiments, the engineered T cells are Jurkat T cells or CTLL-2 Tcells. In some embodiments, the TCS-binding fragment is a CD3-bindingfragment. In some embodiments, the CD3-binding fragment is aCD3ε-binding fragment. In some embodiments, the target antigen isexpressed on the surface of the target cells. In some embodiments, thetarget antigen is CD4, CD8, CD 18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, p150, 95, VLA-4, ICAM-1,VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB) receptor;mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, β7, Tenb2,STEAP, or transmembrane tumor-associated antigens (TAA). In someembodiments, a) the target antigen is HER2 receptor and the target cellis a BT-474 cell, b) the target antigen is HER2 receptor and the targetcell is a SKBR3 cell, c) the target antigen is CD20 and the target cellis a Wil2-S cell, or d) the target antigen is CD79b and the target cellis a BJAB cell. The containers hold the formulations and the labels on,or associated with, the containers may indicate directions for use. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, cultureware, reagents for detecting reporter molecules, andpackage inserts with instructions for use.

In some aspects of the invention, a kit or article of manufacture isprovided comprising a container which holds a composition comprising aTCS (such as a CD3 subunit), or a fragment thereof, conjugated withbiotin, and optionally provides instructions for its use. In someembodiments, the TCS is a CD3 subunit. In some embodiments, the CD3subunit is CD3ε. In some embodiments, the kit further provides a targetantigen, or a fragment thereof. In some embodiments, the target antigenis expressed on the surface of a cell. In some embodiments, the targetantigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34, CD40,CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CTLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). Insome embodiments, the kit further provides a reference TDB assaystandard (a purified TDB of known concentration), and/or a TDB control.The containers hold the formulations and the labels on, or associatedwith, the containers may indicate directions for use. The article ofmanufacture may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,cultureware, reagents for detecting reporter molecules, and packageinserts with instructions for use.

VI. Polypeptides

The polypeptides to be analyzed using the methods described herein aregenerally produced using recombinant techniques. Methods for producingrecombinant proteins are described, e.g., in U.S. Pat. Nos. 5,534,615and 4,816,567, specifically incorporated herein by reference. In someembodiments, the protein of interest is produced in a CHO cell (see,e.g. WO 94/11026). In some embodiments, the polypeptide of interest isproduced in an E. coli cell. See, e.g., U.S. Pat. No. 5,648,237; U.S.Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523, which describestranslation initiation region (TIR) and signal sequences for optimizingexpression and secretion. See also Charlton, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 245-254, describing expression of antibody fragments in E. coli.When using recombinant techniques, the polypeptides can be producedintracellularly, in the periplasmic space, or directly secreted into themedium.

The polypeptides may be recovered from culture medium or from host celllysates. Cells employed in expression of the polypeptides can bedisrupted by various physical or chemical means, such as freeze-thawcycling, sonication, mechanical disruption, or cell lysing agents. Ifthe polypeptide is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, are removed,for example, by centrifugation or ultrafiltration. Carter et al.,Bio/Technology 10: 163-167 (1992) describe a procedure for isolatingpolypeptides which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (OH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the polypeptide issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available polypeptideconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

In some embodiments, the polypeptide in the composition comprising thepolypeptide and one or more contaminants has been purified or partiallypurified prior to analysis by the methods of the invention. For example,the polypeptide of the methods is in an eluent from an affinitychromatography, a cation exchange chromatography, an anion exchangechromatography, a mixed mode chromatography and a hydrophobicinteraction chromatography. In some embodiments, the polypeptide is inan eluent from a Protein A chromatography.

Examples of polypeptides that may be analyzed by the methods of theinvention include but are not limited to immunoglobulins,immunoadhesins, antibodies, enzymes, hormones, fusion proteins,Fc-containing proteins, immunoconjugates, cytokines and interleukins.

(A) Antibodies

In some embodiments of any of the methods described herein, thepolypeptide for use in any of the methods of analyzing polypeptides andformulations comprising the polypeptides by the methods described hereinis an antibody. In some embodiments, the polypeptide is a Tcell-dependent bispecific (TDB) antibody.

Molecular targets for antibodies include CD proteins and their ligands,such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD11a, CD20, CD22,CD34, CD40, CD79α (CD79a), and CD79β (CD79b); (ii) members of the ErbBreceptor family such as the EGF receptor, HER2, HER3 or HER4 receptor;(iii) cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4,ICAM-1, VCAM and αv/β3 integrin, including either alpha or beta subunitsthereof (e.g, anti-CD11a, anti-CD18 or anti-CD11b antibodies); (iv)growth factors such as VEGF; IgE; blood group antigens; flk2/flt3receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3,c-met, tissue factor, β7 etc; (v) cell surface and transmembranetumor-associated antigens (TAA), such as those described in U.S. Pat.No, 7,521,541, and (vi) other targets such as FcRH5, TenB2 and STEAP. Insome embodiments, the antibody is an anti-CD2O/anti-CD3 antibody.Exemplary bispecific antibodies are provided in Table 1.

TABLE 1 Exemplary antibodies CD3 Arm Type Seq 40G5c HVR-HlNYYIH (SEQ ID NO: 1) HVR-H2 WIYPGDGNTKYNEKFKG (SEQ ID NO: 2) HVR-H3DSYSNYYFDY (SEQ ID NO: 3) HVR-Ll KSSQSLLNSRTRKNYLA (SEQ ID NO: 4) HVR-L2WASTRES (SEQ ID NO: 5) HVR-L3 TQSFILRT (SEQ ID NO: 6) 38E4v1 HVR-H1SYYIH (SEQ ID NO: 7) HVR-H2 WIYPENDNTKYNEKFKD (SEQ ID NO: 8) HVR-H3DGYSRYYFDY (SEQ ID NO: 9) HVR-L1 KSSQSLLNSRTRKNYLA (SEQ ID NO: 10)HVR-L2 WTSTRKS (SEQ ID NO: 11) HVR-L3 KQSFILRT (SEQ ID NO: 12) UCHT1v9HVR-H1 GYTMN (SEQ ID NO: 13) HVR-H2 LINPYKGVSTYNQKFKD (SEQ ID NO: 14)HVR-H3 SGYYGDSDWYFDV (SEQ ID NO: 15) HVR-L1 RASQDIRNYLN (SEQ ID NO: 16)HVR-L2 YTSRLES (SEQ ID NO: 17) HVR-L3 QQGNTLPWT (SEQ ID NO: 18) 40G5cVH (hu) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS (SEQ ID NO: 19) VL (hu)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK (SEQ ID NO: 20) 38E4v1 VH (hu)EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS (SEQ ID NO: 21) VL (hu)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWISTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGQGTKVEIK (SEQ ID NO: 22) UCHT1v9 VH (hu)EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKDLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO:23) VL (hu)DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLELK (SEQ ID NO: 24) Target Arm 2h7 v 16 HVR-H1GYTFTSYNMH (SEQ ID NO: 25) CD20 HVR-H2 AIYPGNGDTSYNQKFKG (SEQ ID NO: 26)HVR-H3 VVYYSNSYWYFDV (SEQ ID NO: 27) HVR-L1 RASSSVSYMH (SEQ ID NO: 28)HVR-L2 APSNLAS (SEQ ID N0: 29) HVR-L3 QQWSFNPPT (SEQ ID NO: 30) 2h7 v 16VH EVQLVESGGGLVQPGGSLRLSCAAS GYTFTSYNMH WVRQAPGKGLEWVG AIYPGNGDTSYNQKFKG RFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR VVYYSNSYWYFDV WGQGTLVTVSS (SEQ ID NO: 31) VLDIQMTQSPSSLSASVGDRVTITC RASSSVSYMH WYQQKPGKAPKPLIY APSNLAS GVPSRFSGSGSGTDFILTISSLQPEDFATYYC QQWSFNPPT FGQGTKVEIKR (SEQ ID NO: 32) 4D5 HVR-H1DTYIH (SEQ ID NO: 33) Her2 HVR-H2 RIYPTNGYTRYADSVKG (SEQ ID NO: 34)HVR-H3 WGGDGFYAMDY (SEQ ID NO: 35) HVR-L1 RASQDVNTAVA (SEQ ID NO: 36)HVR-L2 SASFLYS (SEQ ID NO: 37) HVR-L3 QQHYTTPPT (SEQ ID NO: 38) 4D5VH (hu) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 39) VL (hu)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 40)

Other exemplary antibodies include those selected from, and withoutlimitation, anti-estrogen receptor antibody, anti-progesterone receptorantibody, anti-p53 antibody, anti-HER-2/neu antibody, anti-EGFRantibody, anti-cathepsin D antibody, anti-Bcl-2 antibody,anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody,anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoproteinantibody, anti-CEA antibody, anti-retinoblastoma protein antibody,anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody,anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24antibody, anti-CD10 antibody, anti-CD11a antibody, anti-CD11c antibody,anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody,anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody,anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody,anti-CD39 antibody, anti-CD100 antibody, anti-CD95/Fas antibody,anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody,anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratins antibody,anti-vimentin antibody, anti-HPV proteins antibody, anti-kappa lightchains antibody, anti-lambda light chains antibody, anti-melanosomesantibody, anti-prostate specific antigen antibody, anti-S-100 antibody,anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibody,anti-TebB2 antibody, anti-STEAP antibody, and anti-Tn-antigen antibody.

(i) Monoclonal Antibodies

In some embodiments, the antibodies are monoclonal antibodies.Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical and/or bind the same epitope except forpossible variants that arise during production of the monoclonalantibody, such variants generally being present in minor amounts. Thus,the modifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete or polyclonal antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as herein described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the polypeptide used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

In some embodiments, the myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, in some embodiments, the myeloma cell lines aremurine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Insome embodiments, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, polypeptide A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). In some embodiments, the hybridomacells serve as a source of such DNA. Once isolated, the DNA may beplaced into expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese Hamster Ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin polypeptide, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Review articles on recombinantexpression in bacteria of DNA encoding the antibody include Skerra etal., Curr. Opinion in Immunol. 5:256-262 (1993) and Plückthun, Immunol.Revs., 130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature 348:552-554 (1990). Clackson etal., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently joiningto the immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In some embodiments of any of the methods described herein, the antibodyis IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is anIgG monoclonal antibody.

(ii) Humanized Antibodies

In some embodiments, the antibody is a humanized antibody. Methods forhumanizing non-human antibodies have been described in the art. In someembodiments, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et at., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some hypervariableregion residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al. J.Immunol. 151:2296 (1993): Chothia et al., J. Mol. Biol. 196:901 (1987)).Another method uses a particular framework region derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chain variable regions. The same framework may be usedfor several different humanized antibodies (Carter et at., Proc. Natl.Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623(1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, in some embodiments of the methods, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablethat illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

(iii) Human Antibodies

In some embodiments, the antibody is a human antibody. As an alternativeto humanization, human antibodies can be generated. For example, it isnow possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); andU.S. Pat. Nos. 5,591,669; 5,589,369; and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat polypeptide gene of a filamentous bacteriophage, such as M13 or fd,and displayed as functional antibody fragments on the surface of thephage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Thus, the phagemimics some of the properties of the B cell, Phage display can beperformed in a variety of formats; for their review see, e.g., Johnson,Kevin S. and Chiswell, David J., Current Opinion in Structural Biology3:564-571 (1993). Several sources of V-gene segments can be used forphage display. Clackson et al., Nature 352:624-8 (1991) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffithet al., EMBO J. 12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332and 5,573,905.

Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

(iv) Antibody Fragments

In some embodiments, the antibody is an antibody fragment. Varioustechniques have been developed for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992) and Brennan et al., Science229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach.F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody,” e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

In some embodiments, fragments of the antibodies described herein areprovided. In some embodiments, the antibody fragment is an antigenbinding fragment. In some embodiments, the antigen binding fragment isselected from the group consisting of a Fab fragment, a Fab′ fragment, aF(ab′)₂ fragment, a scFv, a Fv, and a diabody.

Bispecfic Antibodies

In some embodiments, the antibody is a bispecific antibody. Bispecificantibodies are antibodies that have binding specificities for at leasttwo different epitopes. Exemplary bispecific antibodies may hind to twodifferent epitopes. Alternatively, a bispecific antibody binding arm maybe combined with an arm that binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fcreceptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) andFcγRIII (CD16) so as to focus cellular defense mechanisms to the cell.Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). In someembodiments, the antibody is a T cell-dependent bispecific (TDB)antibody. In some embodiments, the TDB comprises a target antigenbinding fragment and a T cell receptor binding fragment. In someembodiments, the TDB comprises a target antigen binding fragment and aCD3 binding fragment. In some embodiments, the TDB comprises a targetantigen binding fragment and a CD3ε binding fragment.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. In some embodiments,the fusion is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In someembodiments, the first heavy chain constant region (CH1) containing thesite necessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In some embodiments of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. In some embodiments, the interface comprisesat least a part of the C_(H)3 domain of an antibody constant domain. Inthis method, one or more small amino acid side chains from the interfaceof the first antibody molecule are replaced with larger side chains(e.g. tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a heavychain variable domain (V_(H)) connected to a light chain variable domain(V_(L)) by a linker that is too short to allow pairing between the twodomains on the same chain. Accordingly, the V_(H) and V_(L) domains ofone fragment are forced to pair with the complementary V_(L) and V_(H)domains of another fragment, thereby forming two antigen-binding sites.Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

(i) Multivalent Antibodies

In some embodiments, the antibodies are multivalent antibodies. Amultivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies provided herein can be multivalentantibodies (which are other than of the IgM class) with three or moreantigen binding sites (e.g., tetravalent antibodies), which can bereadily produced by recombinant expression of nucleic acid encoding thepolypeptide chains of the antibody. The multivalent antibody cancomprise a dimerization domain and three or more antigen binding sites.The preferred dimerization domain comprises (or consists of) an Fcregion or a hinge region. In this scenario, the antibody will comprisean Fc region and three or more antigen binding sites amino-terminal tothe Fc region. The preferred multivalent antibody herein comprises (orconsists of) three to about eight, but preferably four, antigen bindingsites. The multivalent antibody comprises at least one polypeptide chain(and preferably two polypeptide chains), wherein the polypeptidechain(s) comprise two or more variable domains. For instance, thepolypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2) n-Fc, wherein VD1is a first variable domain, VD2 is a second variable domain. Fc is onepolypeptide chain of an Fc region, X1 and X2 represent an amino acid orpolypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) maycomprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; orVH-CH1-VH-CH1-Fc region chain. The multivalent antibody hereinpreferably further comprises at least two (and preferably four) lightchain variable domain polypeptides. The multivalent antibody herein may,for instance, comprise from about two to about eight light chainvariable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain. In some embodiments, themultivalent antibody comprises a T cell binding fragment. In someembodiments, the multivalent antibody comprises a T cell receptorbinding fragment. In some embodiments, the multivalent antibodycomprises a CD3 binding fragment. In some embodiments, the multivalentantibody comprises a CD3ε binding fragment.

In some embodiments, the antibody is a multispecific antibody. Exampleof multispecific antibodies include, but are not limited to, an antibodycomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)) where the V_(H)V_(L) unit has polyepitopicspecificity, antibodies having two or more V_(L) and V_(H) domains witheach V_(H)V_(L) unit binding to a different epitope, antibodies havingtwo or more single variable domains with each single variable domainbinding to a different epitope, full length antibodies, antibodyfragments such as Fab, FV, dsFv, scFv, diabodies, bispecific diabodies,triabodies, tri-functional antibodies, antibody fragments that have beenlinked covalently or non-covalently. In some embodiment that antibodyhas polyepitopic specificity; for example, the ability to specificallybind to two or more different epitopes on the same or differenttarget(s). In some embodiments, the antibodies are monospecific; forexample, an antibody that binds only one epitope. According to oneembodiment the multispecific antibody is an IgG antibody that binds toeach epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

(vi) Other Antibody Modifications

It may be desirable to modify the antibody provided herein with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet at., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J., Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement mediated lysis and ADCCcapabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230(1989).

For increasing serum half the serum half life of the antibody, aminoacid alterations can be made in the antibody as described in US2006/0067930, which is hereby incorporated by reference in its entirety.

(B) Polypeptide Variants and Modifications

Amino acid sequence modification(s) of the polypeptides, includingantibodies, described herein may be used in the methods of purifyingpolypeptides (e.g., antibodies) described herein.

(i) Variant Polypeptides

“Polypeptide variant” means a polypeptide, preferably an activepolypeptide, as defined herein having at least about 80% amino acidsequence identity with a full-length native sequence of the polypeptide,a polypeptide sequence lacking the signal peptide, an extracellulardomain of a polypeptide, with or without the signal peptide. Such polypeptide variants include, for instance, polypeptides wherein one or moreamino acid residues are added, or deleted, at the N or C-terminus of thefull-length native amino acid sequence. Ordinarily, a TAT polypeptidevariant will have at least about 80% amino acid sequence identity,alternatively at least about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%amino acid sequence identity, to a full-length native sequencepolypeptide sequence, a polypeptide sequence lacking the signal peptide,an extracellular domain of a polypeptide, with or without the signalpeptide. Optionally, variant polypeptides will have no more than oneconservative amino acid substitution as compared to the nativepolypeptide sequence, alternatively no more than about any of 2, 3, 4,5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared tothe native polypeptide sequence.

The variant polypeptide may be truncated at the N-terminus orC-terminus, or may lack internal residues, for example, when comparedwith a full length native polypeptide. Certain variant polypeptides maylack amino acid residues that are not essential for a desired biologicalactivity. These variant polypeptides with truncations, deletions, andinsertions may be prepared by any of a number of conventionaltechniques. Desired variant polypeptides may be chemically synthesized.Another suitable technique involves isolating and amplifying a nucleicacid fragment encoding a desired variant polypeptide, by polymerasechain reaction (PCR). Oligonucleotides that define the desired terminiof the nucleic acid fragment are employed at the 5′ and 3′ primers inthe PCR. Preferably, variant polypeptides share at least one biologicaland/or immunological activity with the native polypeptide disclosedherein.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme or a polypeptide which increases the serum half-life of theantibody,

For example, it may be desirable to improve the binding affinity and/orother biological properties of the polypeptide. Amino acid sequencevariants of the polypeptide are prepared by introducing appropriatenucleotide changes into the antibody nucleic acid, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the polypeptide. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe polypeptide (e.g., antibody), such as changing the number orposition of glycosylation sites.

Guidance in determining which amino acid residue may be inserted,substituted or deleted without adversely affecting the desired activitymay be found by comparing the sequence of the polypeptide with that ofhomologous known polypeptide molecules and minimizing the number ofamino acid sequence changes made in regions of high homology.

A useful method for identification of certain residues or regions of thepolypeptide (e.g., antibody) that are preferred locations formutagenesis is called “alanine scanning mutagenesis” as described byCunningham and Wells, Science 244:1081-1085 (1989). Here, a residue orgroup of target residues are identified (e.g., charged residues such asArg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably Alanine or Polyalanine) to affectthe interaction of the amino acids with antigen. Those amino acidlocations demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table 2 below under the heading of “exemplary substitutions.” Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “substitutions” in the Table 2, or asfurther described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 2 Original Exemplary Residue Substitutions Substitutions Ala (A)Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys;Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu AsnGlu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile;Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; IleLeu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) ThrThr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; SerPhe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Substantial modifications in the biological properties of thepolypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Aminoacids may be grouped according to similarities in the properties oftheir side chains A. L. Lehninger, Biochemistry second ed., pp. 73-75,Worth Publishers, New York (1975)):

-   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F),    Trp (W), Met (M)-   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),    Asn (N), Gln (Q)-   (3) acidic: Asp (D), Glu (E)-   (4) basic: Lys (K), Arg (R), His(H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln,    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to thepolypeptide to improve its stability (particularly where the antibody isan antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e,g, binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and target. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the polypeptide alters theoriginal glycosylation pattern of the antibody. The polypeptide maycomprise non-amino acid moieties. For example, the polypeptide may beglycosylated. Such glycosylation may occur naturally during expressionof the polypeptide in the host cell or host organism, or may he adeliberate modification arising from human intervention. By altering ismeant deleting one or more carbohydrate moieties found in thepolypeptide, and/or adding one or more glycosylation sites that are notpresent in the polypeptide.

Glycosylation of polypeptide is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the polypeptide is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Removal of carbohydrate moieties present on the polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains,acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

(11) Chimeric Polypeptides

The polypeptide described herein may be modified in a way to formchimeric molecules comprising the polypeptide fused to another,heterologous polypeptide or amino acid sequence. In some embodiments, achimeric molecule comprises a fusion of the polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the polypeptide. The presence of suchepitope-tagged forms of the polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the polypeptide to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag.

In an alternative embodiment, the chimeric molecule may comprise afusion of the polypeptide with an immunoglobulin or a particular regionof an immunoglobulin. A bivalent form of the chimeric molecule isreferred to as an “immunoadhesin.”

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologouspolypeptide with the effector functions of immunoglobulin constantdomains. Structurally, the immunoadhesins comprise a fusion of an aminoacid sequence with the desired binding specificity which is other thanthe antigen recognition and binding site of an antibody (i.e., is“heterologous”), and an immunoglobulin constant domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The Ig fusions preferably include the substitution of a soluble(transmembrane domain deleted or inactivated) form of a polypeptide inplace of at least one variable region within an Ig molecule. In aparticularly preferred embodiment, the immunoglobulin fusion includesthe hinge, CH₂ and CH₃, or the hinge, CH₁, CH₂ and CH₃ regions of anIgG1 molecule.

(iii) Polypeptide Conjugates

The polypeptide for use in polypeptide formulations may be conjugated toa cytotoxic agent such as a chemotherapeutic agent, a growth inhibitoryagent, a toxin (e.g., an enzymatically active toxin of bacterial,fungal, plant, or animal origin, or fragments thereof), or a radioactiveisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such conjugates canbe used. In addition, enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin, and the tricothecenes. A variety of radionuclidesare available for the production of radioconjugated polypeptides.Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re. Conjugates of thepolypeptide and cytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vittea etal., Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the polypeptide.

Conjugates of a polypeptide and one or more small molecule toxins, suchas a calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity,are alsocontemplated herein.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata. Subsequently, it was discovered that certainmicrobes also produce maytansinoids, such as maytansinol and C-3maytansinol esters. Synthetic maytansinol and derivatives and analoguesthereof are also contemplated. There are many linking groups known inthe art for making polypeptide-maytansinoid conjugates, including, forexample, those disclosed in U.S. Pat. No. 5,208,020. The linking groupsinclude disufide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Another conjugate of interest comprises a polypeptide conjugated to oneor more calicheamicin molecules. The calicheamicin family of antibioticsis capable of producing double-stranded DNA breaks at sub-picomolarconcentrations. For the preparation of conjugates of the calicheamicinfamily, see, e.g., U.S. Pat. No. 5,712,374. Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁ ¹,α₂ ¹, α₃ ¹, N-acetyl-γ₁ ¹, PSAG and θ₁ ¹. Another anti-tumor drug thatthe antibody can be conjugated is QFA which is an antifolate. Bothcalicheamicin and QFA have intracellular sites of action and do notreadily cross the plasma membrane. Therefore, cellular uptake of theseagents through polypeptide (e.g., antibody) mediated internalizationgreatly enhances their cytotoxic effects.

Other antitumor agents that can be conjugated to the polypeptidesdescribed herein include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex, as well as esperamicins.

In some embodiments, the polypeptide may be a conjugate between apolypeptide and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase)

In yet another embodiment, the polypeptide (e.g., antibody) may beconjugated to a “receptor” (such streptavidin) for utilization in tumorpre-targeting wherein the polypeptide receptor conjugate is administeredto the patient, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand”(e.g., avidin) which is conjugated to a cytotoxic agent (e.g., aradionucleotide).

In some embodiments, the polypeptide may be conjugated to aprodrug-activating enzyme which converts a prodrug (e.g., a peptidylchemotherapeutic agent) to an active anti-cancer drug. The enzymecomponent of the immunoconjugate includes any enzyme capable of actingon a prodrug in such a way so as to convert it into its more active,cytotoxic form.

Enzymes that are useful include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs into free active drugs.

(iv) Other

Another type of covalent modification of the polypeptide compriseslinking the polypeptide to one of a variety of nonproteinaceouspolymers, e,g., polyethylene glycol, polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol. The polypeptide also may be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,18th edition, Gennaro, A.R., Ed., (1990).

VII. Obtaining Polypeptides for Use in the Formulations and Methods

The polypeptides used in the methods of analysis described herein may beobtained using methods well-known in the art, including therecombination methods. The following sections provide guidance regardingthese methods.

(A) Polynucleotides

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.

Polynucleotides encoding polypeptides may be obtained from any sourceincluding, but not limited to, a cDNA library prepared from tissuebelieved to possess the polypeptide mRNA and to express it at adetectable level. Accordingly, polynucleotides encoding polypeptide canbe conveniently obtained from a cDNA library prepared from human tissue.The polypeptide-encoding gene may also be obtained from a genomiclibrary or by known synthetic procedures (e.g., automated nucleic acidsynthesis).

For example, the polynucleotide may encode an entire immunoglobulinmolecule chain, such as a light chain or a heavy chain. A complete heavychain includes not only a heavy chain variable region (V_(H)) but also aheavy chain constant region (C_(H)), which typically will comprise threeconstant domains: C_(H)1, C_(H)2 and C_(H)3; and a “hinge” region. Insome situations, the presence of a constant region is desirable. In someembodiments, the polynucleotide encodes one or more immunoglobulinmolecule chains of a TDB.

Other polypeptides which may be encoded by the polynucleotide includeantigen-binding antibody fragments such as single domain antibodies(“dAbs”), Fv, scFv, Fab′ and F(ab)₂ and “minibodies.” Minibodies are(typically) bivalent antibody fragments from which the C_(H)1 and C_(K)or C_(L) domain has been excised. As minibodies are smaller thanconventional antibodies they should achieve better tissue penetration inclinical/diagnostic use, but being bivalent they should retain higherbinding affinity than monovalent antibody fragments, such as dAbs.Accordingly, unless the context dictates otherwise, the term “antibody”as used herein encompasses not only whole antibody molecules but alsoantigen-binding antibody fragments of the type discussed above.Preferably each framework region present in the encoded polypeptide willcomprise at least one amino acid substitution relative to thecorresponding human acceptor framework. Thus, for example, the frameworkregions may comprise, in total, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acidsubstitutions relative to the acceptor framework regions.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Further details of the invention are illustrated by the followingnon-limiting Examples. The disclosures of all references in thespecification are expressly incorporated herein by reference.

EXAMPLES

The examples below are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway. The following examples and detailed description are offered by wayof illustration and not by way of limitation.

Example 1 T Cell Activation Assay

A T cell activation assay has been developed to determine the potencyand specificity of a T Cell Dependent Bispecific (TDB) antibody foractivating T cells in the presence of target cells. See FIG. 2 for anexemplary schematic representation. As TDBs are bivalent and bispecific,with one arm specific for a TCR complex subunit and the other specificfor a target antigen, cross-linking of TCRs leading to T cell activationcan only occur when both the target cell and the T cell are bound by theTDB. TCR mediated cross-linking by anti-CD3-containing TDBs activates Tcell signal transduction cascades leading to the phosphorylation andnuclear localization of transcription factors, including NFAT and NFκB,resulting in the transcriptional induction of target genes such ascytokines or cell killing agents such as Fas, Granzyme B and Perforins(Brown, W M, 2006, Curr Opin Investig Drugs 7:381-388; Ferran, C et al.,1993 Exp Nephrol 1:83-89; Shannon, M F et al., 1995, J. Leukoc. Biol.57:767-773; Shapiro, 1998; Pardo, J, et al., 2003, Int Immunol.,15(12):1441-1450). Reporter genes, such as firefly luciferase, under thetranscriptional control of AP1, MAT, or NFκB, have been used to monitorTCR activation of signaling pathways and T cell activation (Shannon, M Fet al., 1995, J. Leukoc. Biol. 57:767-773; Shapiro, 1998). To evaluateif TDBs can activate T cells in vitro, Jurkat T cells (DSMZ, ACC 282)were infected with recombinant TCR-responsive reporter gene lentiviralstocks (AP1-Luciferase, NFAT-Luciferase, or NFκB-Luciferase) and stablepools of reporter T cells were isolated. To initially assess thesuitability of the reporter T cells for assaying activation, they weretreated with purified Anti-CD3 homodimer, which can cross-link TCRreceptors in the absence of target cells, at 10 μg/mL for 4 hours.Jurkat/AP1Luciferase, Jurkat/NFATLuciferase, and Jurkat/NFκBLuciferasestable pools showed a dose-dependent induction of luciferase uponstimulation with purified Anti-CD3 homodimer. Luminescence responses(luciferase reporter gene activity) were plotted, with the highestresponse observed from the Jurkat/NFκBluciferase stable pool (FIG. 3A).Jurkat/NFκBLuciferase stable clones isolated by limiting dilution werescreened for their response to 10 μg/mL of purified Anti-CD3 homodimer.Jurkat T cell NFκBLuciferase pools demonstrated the highest response toAnti-CD3 homodimer compared to other TCR-response elements, and wereselected for further investigation (FIG. 39).

To determine the relative response of this clone to either αCD20/αCD3TDB or to anti-CD3 homodimer, the Jurkat/NFκBLuciferase clone 2 cellline was treated with increasing concentrations of either αCD20/αCD3 TDBor anti-CD3 homodimer in the presence of a CD20 expressing target cellline, and luciferase activity was plotted (FIG. 4A). The cells werestimulated with αCD20/αCD3 TDB or a CD3 homodimer for 4 hours in RPMI1640 medium supplemented with 10% Fetal Bovine Serum. PurifiedαCD20/αCD3 TDB was 1000-fold more active than purified anti-CD3homodimer in the presence of co-stimulatory target cells. In the absenceof target cells, αCD20/αCD3 TDB did not result in T cell activation ateven high levels of the TDB, as measured by NFκB-dependent activation ofluciferase transcription in this cell line, indicating the specificityof the assay for detecting simultaneous binding of the TDB to target andeffector cells (FIG. 49). The T cell activation responses observed forthe engineered Jurkat/NFκBLuciferase clone 2 reporter gene cell line iscomparable to that observed using human T cells isolated from donorPeripheral Blood Mononuclear Cells (PBMCs) using other measures of Tcell activation, indicating that the use of a reporter gene to monitor Tcell activation response is comparable (Table 3). TheJurkat/NFκBluciferase clone 2 cell line (Jurkat-NFκBLuc), was used todevelop and optimize a cell-based assay method for the detection ofTDB-mediated T cell activation.

TABLE 3 Anti-CD3 homodimer αCD20/αCD3 TDB (EC₅₀) in absence of (EC₅₀) inpresence target cells of target cells Human PBMC 526 ng/mL 5.5 ng/mL(CD69⁺/CD25⁺) Donor 1 Human PBMC 169 ng/mL 4.4 ng/mL (CD69⁺/CD25⁺) Donor2 Jurkat/NFκBLuc 210 ng/mL 1.3 ng/mL

Example 2 Quantitative Method to Detect TDB-Mediated T Cell Activation

A sensitive and quantitative platform TDB cell-based assay to determinethe potency of anti-CD3 containing TDBs by measuring the induction of Tcell activation in the presence of target cells has been developed. TheTDB T cell activation assay detects activation of T cells by a TDB inthe presence of target cells by measuring TCR cross-linking-inducedactivation of the Rel/NFκB signaling pathway using an engineered T cellreporter gene cell line, Jurkat-NFκBLuc. Activated NFκB translocates tothe nucleus, binds to the 8 NFκB response elements in the syntheticpromoter and drives the transcription of luciferase.

In the assay, dilutions of Anti-CD20/CD3 (or Anti-HER2/CD3 orAnti-CD79b/CD3) assay standard, control, and test samples were preparedand 50 μL was added to 96 well assay plates. Target cells (Wil2-S,BT-474 or SKBR3, and BJAB cells for αCD20/CD3, αHER2/CD3, andαCD79b/CD3, respectively) and JurkatNFkB reporter cells were thenprepared, using either ready-to-use (R-to-U) frozen cells or culturedcells following assessment that frozen cells are comparable to freshcultured cells. Equal volumes of 4.0×10⁵ cells/mL of target cell diluentand 1.6×10⁶ cells/mL JurkatNFkB cell diluent were combined to prepare acell mixture with a target:effector (T:E) cell ratio of 1:4. 50 μL ofthe mixed target and JurkatNFkBLuc cells was added to the each TDBdilution in the assay plate. The same T:E ratio was used for αHER2/CD3and αCD79b/CD3 cell based assays as well for the assays including thereference and control TDBs. Following target cell conjugation to theJurkatNFkBLuc T cell reporter cell line by the TDB, activated NFκBtranslocates to the nucleus, binds to the 8 NFκB response elements inthe synthetic promoter and drives the transcription of luciferase. After4-5 hours of assay incubation, the amount of luciferase activity inducedby each sample was measured using a luminescence plate reader (FIG. 5).The relative potency of the control and the test samples was determinedfrom a standard curve of luminescence generated from the TDB referencestandard using 4P analysis as follows:

-   -   Use a 4-parameter logistic curve-fitting program to generate        separate curves for standard, control and sample(s).    -   The equation is:

y=((A−D)/(1+(x/C)̂B))+D

-   -   Where: x=the independent variable        -   A=Zero Dose Response (Lower asymptote=LA)        -   B=Slope        -   C=EC50, ng/mL        -   D=Maximum Dose Response (Upper asymptote=UA)    -   Determine the fold response for the standard curve (ST) and each        test article (TA) (control or sample(s)) curve as follows:        -   Amplitude of response=UA/LA    -   Report the value.    -   Check for similarity between the ST and each TA curve as        follows:    -   Test for parallelism. Determine the Slope Ratio using the        following equation:

$\frac{{\left( {D_{TA} - A_{TA}} \right) \times B_{TA}}}{{\left( {D_{ST} - A_{ST}} \right) \times B_{ST}}}$

-   -   Determine the Lower asymptote percent difference (LAD) using the        following equation:

${\frac{{LA}_{TA} - {LA}_{ST}}{{UA}_{ST} - {LA}_{ST}} \times 100}$

-   -   Determine the Upper asymptote percent difference (UAD) using the        following equation:

${\frac{{UA}_{TA} - {UA}_{ST}}{{UA}_{ST} - {LA}_{ST}} \times 100}$

-   -   Calculate the potency of the test article using a constrained        4-P parallel curve.    -   For the ST and TA generate constrained curves using the        following equations:

$y_{ST} = {D + \frac{A - D}{1 + \left( \frac{x}{C_{ST}} \right)^{B}}}$$y_{TA} = {D + \frac{A - D}{1 + \left( \frac{\rho \; x}{C_{ST}} \right)^{B}}}$

-   -   Where: x=the independent variable        -   A=Common lower asymptote        -   B=Common slope        -   CST=Standard EC50 value        -   D=Common upper asymptote    -   ρ=relative potency of test article (the relative potency is the        ratio of EC50 of ST over EC50 of TA)    -   Calculate the potency, expressed as % Relative Potency, for the        control and sample(s):        -   Potency=ρ×Activity of Reference Material

CD69 (C-type lectin protein) and CD25 (IL-2 receptor) are markers of Tcell activation (Shipkova M, 2012, Clin. Chim. Acta. 413:1338-49 andZiegler S F, et al., 1994, Stern Cells 12(5): 465-465), and theirinduction on the surface of T cells 24 hours following addition of theαCD20/CD3 TDB was evaluated by flow cytometry. CD69 and CD25 cellsurface expression increased in a dose-dependent manner in response toincubation with the αCD20/αCD3 TDB (FIGS. 6A and 6B), and thedose-response curve correlated well with that obtained by measuring theluciferase signal from the JurkatNFkBLuc T cell reporter cell line (FIG.7), demonstrating that the luciferase measurement is a relevant readoutof T cell activation in the TDB cell-based assay using JurkatNFkBLuccells.

The amount of simultaneous binding of the TDB with its targets wasassessed using an ELISA-based bridging binding assay. See FIG. 8 for anexemplary schematic representation. The bridging of a TCR complexsubunit and the extracellular domain (ECD) of the target antigen by aTDB is an essential interaction representing the mechanism of action ofthe TDB. The assay was used to detect the different affinities ofanti-HER2/CD3 TDB variants (FIG. 9), and anti-HER2/CD3 samples subjectedto thermal stress conditions (2 wks and 4 wks at 40° C., see FIG. 10A).In the TDB bridging binding assay for anti-HER2/CD3, HER2 ECD (CR#156)was used as coating material on the plate. After 16-72 hours, the platewas washed and then αHER2/CD3 TDB was incubated for 1-2 hour in assaydiluent. After washing, biotinylated CD3ε peptide was incubated for 1-2hours, followed by Strep-HRP incubation. After a final wash, the amountof HRP conjugated to the plate was measured using a detection agent. Astrong correlation (R²=0.9976) between the bridging binding assay andthe TDB cell-based assay was observed (FIG. 10B), further supporting theuse of the TDB cell-based assay as a measure of TDB potency.

Bridging Assay

Materials

Coat material: HER2 ECD (CR#156)

Coating buffer: Dulbecco's Phosphate Buffered Saline without CaCl₂ andMgCl₂, Gibco Cat. No. 14190

Assay Diluent/Detection Dilution Buffer: PBS+0.5% BSA+0.05% PS20

Wash buffer:PBS+0.05% PS20

Detection: Strep-HRP

Coat plates

1. Dilute coat reagent to 4 μg/mL in coating butler

2. Coat all 96 wells with 100 μL.

3. Seal with plate sealer.

4. Incubate at 2-8° C. 16-72 hours.

Assay Procedure

-   -   1. Wash the plates 6 times with wash buffer using the wash        program “Auto” (i.e., Run two cycles of Auto program).    -   2. Block the plates using 200 μL of AD per well. Seal and        incubate 1-2 hours at 25° C. with shaking.    -   3. Prepare αHER2 TDB Ab in AD.        -   4. After blocking, Wash the plate 6 times as step 1    -   5. Add 100 uL of diluted αHER2 TDB to each well. Incubated 1        hours+10 min at 25° C. with shaking.    -   6. Repeat Step 1.    -   7. Prepare CD3e peptide (16 mer) with final concentration: 1        μg/mL of CD3e peptide    -   8. Add 100 μL to each well. Incubate for 1 hour    -   9. Repeat Step 1.        -   10. Add Strep-HRP, final concentration of 2 ng/mL, and            incubate at 25C for 1 hour    -   11. Repeat Step 1.    -   12. Add 100 μL Sure Blue Reserve to wells. Develop until optimal        color development before stopping reaction with 100 μL 0.6 N        Sulfuric Acid.    -   15. Read OD 450/650

Example 3 Analysis of Anti-FcRH5/Anti-CD3 Antibody

The assays described in Example 2 were used to measure the potency ofand anti-FcRH5/anti-CD3 TBD. In the assay, dilutions of anti-FcRH5/CD3assay standard, control, and test samples were prepared and 50 μL wasadded to 96 well assay plates. Target cells (FcRH5-expressing EJM cells)and JurkatNFkB reporter cells were then prepared, using eitherready-to-use (R-to-U) frozen cells or cultured cells followingassessment that frozen cells are comparable to fresh cultured cells.Equal volumes of 4.0×10⁵ cells/mL of target cell diluent and 1.6×10°cells/mL JurkatNFkB cell diluent were combined to prepare a cell Mixturewith a target:effector (T:E) cell ratio of 1:4. 50 μL of the mixedtarget and JurkatNFkBLuc cells was added to the each TDB dilution in theassay plate. The same T:E ratio was used for the reference and controlTDBs. After 4-5 hours of assay incubation, the amount of luciferaseactivity induced by each sample was measured using a luminescence platereader (FIG. 11). The relative potency of the control and the testsamples was determined from a standard curve of luminescence generatedfrom the TDB reference standard using 4P analysis as follows describedin Example 2.

The amount of simultaneous binding of the anti-FcRH5/CD3 TDB with itstargets was assessed using an ELISA-based bridging binding assay asdescribed in Example 2. In the TDB bridging binding assay foranti-FcRH5/CD3, domain 9 FcRH5 ECD was used as coating material on theplate. After 16-72 hours, the plate was washed and then αFcRH5/CD3 TDBwas incubated for 1-2 hour in assay diluent. After washing, biotinylatedCD3ε peptide was incubated for 1-2 hours, followed by Strep-HRPincubation. After a final wash, the amount of HRP conjugated to theplate was measured using a detection agent (FIG. 12).

What is claimed is:
 1. A method for detecting a T cell dependentbispecific antibody (TDB) in a composition, wherein the bispecificantibody comprises a target antigen binding fragment and a CD3 bindingfragment, the method comprising contacting a population of T cells andtarget cells with the composition, wherein the T cells comprise nucleicacid encoding a reporter operably linked to a response element that isresponsive to T cell activation, and wherein the target cells expressthe target antigen, wherein expression of the reporter indicates thepresence of TDBs.
 2. The method of claim 1, wherein the reporter is aluciferase, a fluorescent protein, an alkaline phosphatase, a betalactamase, or a beta galactosidase.
 3. The method of claim 2, whereinthe luciferase is a firefly luciferase, Renilla luciferase, or ananoluciferase.
 4. The method of any one of claims 1-3, wherein theresponse element that is responsive to T cell activation is an NFATpromoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3promoter, a STAT5 promoter or an IRF promoter.
 5. The method of claim 4,wherein the response element that is responsive to T cell activationcomprises T cell activation responsive elements from any one or more ofNFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.
 6. The method of any oneof claims 1-5, wherein the population of T cells is population of CD4⁺ Tcells or CD8⁺ T
 7. The method of any one of claims claim 1-5, whereinthe population of T cells is population of Jurkat T cells or CTLL-2cells.
 8. The method of any one of claims 1-7, wherein the targetantigen is expressed on the surface of the target cell.
 9. The method ofany one of claims 1-8, wherein the target antigen is CD4, CD8, CD18,CD19, CD11a, CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β(CD79b), EGF receptor, HER2 receptor, HER3 receptor, HER4 receptor,FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4, ICAM 1, VCAM, αv/β3 integrin,VEGF, flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4;protein C, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA).
 10. The method of any one of claims1-9, wherein a) the target antigen is HER2 receptor and the target cellis a BT-474 cell, b) the target antigen is HER2 receptor and the targetcell is a SKBR3 cell, c) the target antigen is CD20 and the target cellis a Wil2-S cell, or d) the target antigen is CD79b and the target cellis a BJAB cell.
 11. The method of any one of claims 1-10, wherein theratio of T cells to target cells in the population of cells is about1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7,about 1:8, about 1:9 or about 1:10.
 12. The method of any one of claims1-11, wherein the ratio of T cells to target cells in the population ofcells is about 1:4.
 13. The method of any one of claims 1-12, whereinthe population of cells ranges from about 1×10³ to about 1×10⁶.
 14. Themethod of any one of claims 1-12, wherein the population of cells isabout 1×10⁴ to about 5×10⁴.
 15. The method of any one of claims 1-14,wherein population of T cells is contacted with a composition comprisingthe TDB at a concentration ranging from 0.01 ng/mL to 50 ng/mL.
 16. Themethod of any one of claims 1-15, wherein the reporter is detected afterany one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20 or 24 hours aftercontacting the cells with the composition.
 17. Method for quantitatingthe amount of TDB an in a composition, wherein the TDB comprises atarget antigen binding fragment and a CD3 binding fragment, the methodcomprising contacting a population of T cells and target cells with thecomposition at one or more concentrations of the composition, whereinthe T cells comprise nucleic acid encoding a reporter operably linked toa response element that is responsive to T cell activation, and whereinthe target cells express the target antigen; correlating the expressionof the reporter as a function of antibody concentration with a standardcurve generated by contacting the population of cells and target cellswith different concentrations of purified TDB.
 18. The method of claim17, wherein the reporter is a luciferase, a fluorescent protein, analkaline phosphatase, beta lactamase, or a beta galactosidase.
 19. Themethod of claim 18, wherein the luciferase is a firefly luciferase, aRenilla luciferase, or a nanoluciferase.
 20. The method of any one ofclaims 17-19, wherein the response element that is responsive to T cellactivation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, aFOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter.21. The method of claim 20, wherein the response element that isresponsive to T cell activation comprises T cell activation responsiveelements from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5and IRF.
 22. The method of any one of claims 17-21, wherein thepopulation of T cells is population of CDC4⁺ T cells or CD8⁺ T cells.23. The method of any one of claims claim 17-22, wherein the populationof T cells is population of Jurkat T cells or CTLL-2 T cells.
 24. Themethod of any one of claims 17-23, wherein the target antigen isexpressed on the surface of the target cell.
 25. The method of any oneof claims 17-24, wherein the target antigen is CD4, CD8, CD18, CD19,CD11a, CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGFreceptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CLL1,LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3 integrin VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA).
 26. The method of any one of claims17-25, wherein a) the target antigen is HER2 receptor and the targetcell is a BT-474 cell, b) the target antigen is HER2 receptor and thetarget cell is a SKBR3 cell, c) the target antigen is CD20 and thetarget cell is a Wil2-S cell, or d) the target antigen is CD79b and thetarget cell is a BJAB cell.
 27. The method of any one of claims 17-26,wherein the ratio of T cells to target cells in the population of cellsis about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6,about 1:7, about 1:8, about 1:9 or about 1:10.
 28. The method of any oneof claims 17-27, wherein the ratio of T cells to target cells in thepopulation of cells is about 1:4.
 29. The method of any one of claims17-28, wherein the population of cells ranges from about 1×10³ to about1×10⁶.
 30. The method of any one of claims 17-29, wherein the populationof cells is about 1×10⁴ to about 5×10⁴.
 31. The method of any one ofclaims 17-30, wherein population of T cells is contacted with acomposition comprising the TDB at a concentration ranging from about0.01 ng/mL, to about 100 ng/mL.
 32. The method of any one of claims17-31, wherein the standard curve is generated by contacting the T cellswith different concentrations of purified anti-CD3 antibody ranging fromabout 0.01 ng/mL to about 100 ng/mL.
 33. The method of any one of claims17-32, wherein the reporter is detected after any one or more of 1, 2,3, 4, 5, 6, 7, 8, 12, 16, 20 or 24 hours after contacting the cells withthe composition.
 34. Method for determining the specificity of a TDB,wherein the TDB comprises a target antigen binding fragment and a CD3binding fragment, the method comprising a) contacting a population of Tcells and test cells with the TDB, wherein the T cells comprise nucleicacid encoding a reporter operably linked to a response element that isresponsive to T cell activation, and wherein the test cells do notexpress the target antigen; b) contacting a population of T cells andtest cells with the TDB, wherein the T cells comprise nucleic acidencoding a reporter operably linked to a response element that isresponsive to T cell activation, and wherein the test cells do notexpress the target antigen; comparing expression of the reporter in thepresence of the test cell in part a) with expression of the reporter inthe presence of target cells in part b), wherein the ratio of expressionof the reporter of the test cells to the target cells is indicative ofthe specificity of the TDB.
 35. The method of claim 34, wherein thereporter is a luciferase, a fluorescent protein, an alkalinephosphatase, beta lactamase, or a beta galactosidase.
 36. The method ofclaim 35, wherein the luciferase is a firefly luciferase, a Renillaluciferase, or a nanoluciferase.
 37. The method of any one of claims 34,wherein the response element that is responsive to cell activation is anNFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, aSTAT3 promoter, a. STAT5 promoter or an IRF promoter.
 38. The method ofclaim 37, wherein the response element that is responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.
 39. The methodof any one of claims 34-38, wherein the population of T cells ispopulation of CD4⁺ T cells or CD8⁺ T cells.
 40. The method of any one ofclaims 34-38, wherein the population of T cells is population of JurkatT cells or CTLL-2 T cells.
 41. The method of any one of claims 34-40,wherein the target antigen is expressed on the surface of the targetcell.
 42. The method of any one of claims 34-41, wherein the targetantigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34, CD40,CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAD, or transmembrane tumor-associated antigens (TAA). 43.The method of any one of claims 34-42, wherein a) the target antigen isHER2 receptor and the target cell is a BT-474 cell, b) the targetantigen is HER2 receptor and the target cell is a SKBR3 cell, c) thetarget antigen is CD2) and the target cell is a Wil2-S cell, or d) thetarget antigen is CD79b and the target cell is a BJAB cell.
 44. Themethod of any one of claims 34-43, wherein the ratio of T cells to testcells in the population of cells of step a) and/or the ratio of T cellsto target cells in the population of cells of step b) is about 1:1,about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about1:8, about 1:9 or about 1:10.
 45. The method of any one of claims 34-44,wherein the ratio of T cells to test cells in the population of cells ofstep a) and/or the ratio of T cells to target cells in the population ofcells or step b) is about 1:4.
 46. The method of any one of claims34-45, wherein the population of cells of steps a and/or b) ranges fromabout 1×10³ to about 1×10⁶.
 47. The method of any one of claims 34-46,wherein the population of cells of steps a and/or b) ranges from about1×10⁴ to about 5×10⁴.
 48. The method of any one of claims 34-47, whereinpopulation of T cells and test cells of step a) and the population of Tcells and target cells of step b) are contacted with a compositioncomprising the TDB at a concentration ranging from about 0.01 ng/mL toabout 100 ng/mL.
 49. The method of any one of claims 34-48, wherein thereporter is detected after any one or more of 1, 2, 3, 4, 5, 6, 7, 8,12, 16, 20 or 24 hours after contacting the cells with the composition.50. A kit for the detection of TDB in a composition comprising abispecific antibody where the bispecific antibody comprises a targetantigen binding fragment and a CD3 binding fragment, wherein the kitcomprises an engineered T cell comprising a reporter operably linked toa response element that is responsive to T cell activation.
 51. The kitof claim 50, further comprising a TDB assay standard and/or a TDBcontrol.
 52. The kit of claim 50 or 51, further comprising target cellswhich express the target antigen.
 53. The kit of any one of claims50-52, wherein the reporter is a luciferase, a fluorescent protein, analkaline phosphatase, a beta lactamase, or a beta galactosidase.
 54. Thekit of claim 53, wherein the luciferase is a firefly luciferase, Renillaluciferase, or a nanoluciferase.
 55. The kit of any one of claims 50-54,wherein the response element that is responsive to T cell activation isan NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, aSTAT3 promoter, a STAT5 promoter or an IRF promoter.
 56. The kit ofclaim 55, wherein the response element that is responsive to T cellactivation comprises T cell activation responsive elements from any oneor more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.
 57. The kit ofany one of claims 50-56, wherein the population of T cells is populationof CD4⁺ T cells or CD8⁺ T cells.
 58. The kit of any one of claims claim50-57, wherein the population of T cells is population of Jurkat T cellsor CTLL-2 T cells.
 59. The kit of any one of claims 50-58, wherein thetarget antigen is expressed on the surface of a target cell.
 60. The kitof any one of claims 50-59, wherein the target antigen is CD4, CD8,CD18, CD19, CD11a, CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β(CD79b), EGF receptor, HER2 receptor, HER3 receptor, HER4 receptor,FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, αv/β3 integrin, VEGF,flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembranetumor-associated antigens (TAA).
 61. The kit of any one of claims 52-60,wherein a) the target antigen is HER2 receptor and the target cell is aBT-474 cell, b) the target antigen is HER2 receptor and the target cellis a SKBR3 cell, c) the target antigen is CD20 and the target cell is aWil2-S cell, or d) the target antigen is CD79b and the target cell is aBJAB cell,
 62. A kit for use in the method of any one of claims 1-49.63. A method for determining if a population of test cells expresses atarget antigen, the method comprising a) contacting the population oftest cells with a population of T cells, wherein the T cells comprisenucleic acid encoding a reporter operably linked to a response elementthat is responsive to T cell activation, b) contacting the population ofT cells and test cells with the TDB, wherein the TDB comprises a targetantigen binding fragment and a CD3 binding fragment, wherein expressionof the reporter indicates the presence of the target antigen expressedby the test cell.
 64. The method of claim 63, wherein the reporter is aluciferase, a fluorescent protein, an alkaline phosphatase, betalactamase, or a beta galactosidase.
 65. The method of claim 64, whereinthe luciferase is a firefly luciferase, a Renilla luciferase, or ananoluciferase.
 66. The method of any one of claims 65, wherein theresponse element that is responsive to T cell activation is an NFATpromoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3promoter, a STAT5 promoter or an IRF promoter.
 67. The method of claim66, wherein the response element that is responsive to T cell activationcomprises T cell activation responsive elements from any one or more ofNFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.
 68. The method of any oneof claims 63-67, wherein the population of T cells is population of CD4⁺T cells or CD8⁺ T
 69. The method of any one of claims claim 63-68,wherein the population of T cells is population of Jurkat T cells orCTLL-2 T cells.
 70. The method of any one of claims 63-69, wherein thetarget antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34,CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Macl1, p150, 95, VLA-4,ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor,β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). 71.The method of any one of claims 63-70, wherein the test cells are tumorcells, immune cells or vascular cells.
 72. The method of any one ofclaims 63-71, wherein the test cells do no comprise T cells.
 73. Themethod of any one of claims 63-72, wherein the ratio of T cells to testcells is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9 or about 1:10.
 74. The method ofany one of claims 63-73, wherein the ratio of T cells to test cells isabout 1:4.
 75. The method of any one of claims 63-74, wherein thepopulation of test cells and T cells ranges from about 1×10³ to about1×10⁶.
 76. The method of any one of claims 63-75, wherein the populationof test cells and T cells is about 1×10⁴ to about 5×10⁴.
 77. The methodof any one of claims 63-76, wherein population of test cells and T cellsis contacted with the TDB at a concentration ranging from about 0.01ng/mL to about 100 ng/mL.
 78. The method of any one of claims 63-77,wherein the reporter is detected after any one or more of 1, 2, 3, 4, 5,6, 7, 8, 12, 16, 20 or 24 hours after contacting the cells with thecomposition.